WorldWideScience

Sample records for kappa phage receptor

  1. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.......-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable...... cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does...

  2. Primary isolation strain determines both phage type and receptors recognised by Campylobacter jejuni bacteriophages.

    Science.gov (United States)

    Sørensen, Martine C Holst; Gencay, Yilmaz Emre; Birk, Tina; Baldvinsson, Signe Berg; Jäckel, Claudia; Hammerl, Jens A; Vegge, Christina S; Neve, Horst; Brøndsted, Lone

    2015-01-01

    In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.

  3. Primary isolation strain determines both phage type and receptors recognised by Campylobacter jejuni bacteriophages.

    Directory of Open Access Journals (Sweden)

    Martine C Holst Sørensen

    Full Text Available In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb, host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220 as well as receptors (CPS or flagella recognised by the isolated phages.

  4. Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

    DEFF Research Database (Denmark)

    Sørensen, Martine C. Holst; Gencay, Yilmaz Emre; Birk, Tina

    2015-01-01

    In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated...... using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We...... therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages...

  5. Kappa opioid receptors stimulate phosphoinositide turnover in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Periyasamy, S.; Hoss, W. (Univ. of Toledo, OH (USA))

    1990-01-01

    The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The {kappa}-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other {kappa}-agonists Dynorphin-A (1-13) amide, and its protected analog D(Ala){sup 2}-dynorphin-A (1-13) amide also produced a significant increase in the formation of ({sup 3}H)-IP's, whereas the {mu}-selective agonists (D-Ala{sup 2}-N-Me-Phe{sup 4}-Gly{sup 5}-ol)-enkephalin and morphine and the {delta}-selective agonist (D-Pen{sup 2,5})-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the {kappa}-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medullar. The results indicate that brain {kappa}- but neither {mu}- nor {delta}- receptors are coupled to the PI turnover response.

  6. Synaptic localization of. kappa. opioid receptors in guinea pig neostriatum

    Energy Technology Data Exchange (ETDEWEB)

    Jomary, C.; Beaudet, A. (McGill Univ., Montreal, Quebec (Canada)); Gairin, J.E. (Centre National de la Recherche Scientifique, Toulouse (France))

    1992-01-15

    Distribution of {kappa} opioid receptors was examined by EM radioautography in sections of guinea pig neostriatum with the selective {sup 125}I-labeled dynorphin analog (D-Pro{sup 10})dynorphin-(1-11). Most specifically labeled binding sites were found by probability circle analysis to be associated with neuronal membrane appositions. Because of limitations in resolution of the method, the radioactive sources could not be ascribed directly to either one of the apposed plasma membranes. Nevertheless, three lines of evidence favored a predominant association of ligand with dendrites of intrinsic striatal neurons: (1) the high frequency with which labeled interfaces implicated a dendrite, (2) the enrichment of dendrodendritic interfaces, and (3) the occurrence of dendritic profiles labeled at several contact points along their plasma membranes. A small proportion of labeled sites was associated with axo-axonic interfaces, which may subserve the {kappa} opioid-induced regulation of presynaptic dopamine and acetylcholine release documented in guinea pig neostriatum. These results support the hypothesis that in mammalian brain {kappa} opioid receptors are conformationally and functionally distinct from {mu} and {delta} types.

  7. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Directory of Open Access Journals (Sweden)

    E. Teodorov

    2012-10-01

    Full Text Available The periaqueductal gray (PAG has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc or 0.9% saline (up to 1 mL/kg and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05 because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR. A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05 and lactating female rats (P < 0.01, with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in

  8. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  9. Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

    Directory of Open Access Journals (Sweden)

    Andrey A Filippov

    Full Text Available BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

  10. Does the kappa opioid receptor system contribute to pain aversion?

    Directory of Open Access Journals (Sweden)

    Catherine M Cahill

    2014-11-01

    Full Text Available The kappa opioid receptor (KOR and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain.

  11. Prefrontal Cortical Kappa Opioid Receptors Attenuate Responses to Amygdala Inputs.

    Science.gov (United States)

    Tejeda, Hugo A; Hanks, Ashley N; Scott, Liam; Mejias-Aponte, Carlos; Hughes, Zoë A; O'Donnell, Patricio

    2015-12-01

    Kappa opioid receptors (KORs) have been implicated in anxiety and stress, conditions that involve activation of projections from the basolateral amygdala (BLA) to the medial prefrontal cortex (mPFC). Although KORs have been studied in several brain regions, their role on mPFC physiology and on BLA projections to the mPFC remains unclear. Here, we explored whether KORs modify synaptic inputs from the BLA to the mPFC using in vivo electrophysiological recordings with electrical and optogenetic stimulation. Systemic administration of the KOR agonist U69,593 inhibited BLA-evoked synaptic responses in the mPFC without altering hippocampus-evoked responses. Intra-mPFC U69,593 inhibited electrical and optogenetic BLA-evoked synaptic responses, an effect blocked by the KOR antagonist nor-BNI. Bilateral intra-mPFC injection of the KOR antagonist nor-BNI increased center time in the open field test, suggesting an anxiolytic effect. The data demonstrate that mPFC KORs negatively regulate glutamatergic synaptic transmission in the BLA-mPFC pathway and anxiety-like behavior. These findings provide a framework whereby KOR signaling during stress and anxiety can regulate the flow of emotional state information from the BLA to the mPFC.

  12. The dynamic relationship between mu and kappa opioid receptors in body temperature regulation.

    Science.gov (United States)

    Chen, Xiaohong; McClatchy, Daniel B; Geller, Ellen B; Tallarida, Ronald J; Adler, Martin W

    2005-12-12

    Previous studies demonstrated that intracerebroventricular (icv) injection of a kappa opioid receptor agonist decreased, and a mu agonist increased, body temperature (Tb) in rats. A dose-response study with the selective kappa antagonist nor-binaltorphimine (nor-BNI) showed that a low dose (1.25 nmol, icv) alone had no effect, although a high dose (25 nmol, icv) increased Tb. It was hypothesized that the hyperthermia induced by nor-BNI was the result of the antagonist blocking the kappa opioid receptor and releasing its inhibition of mu opioid receptor activity. To determine whether the Tb increase caused by nor-BNI was a mu receptor-mediated effect, we administered the selective mu antagonist CTAP (1.25 nmol, icv) 15 min after nor-BNI (25 nmol, icv) and measured rectal Tb in unrestrained rats. CTAP significantly antagonized the Tb increase induced by icv injection of nor-BNI. Injection of 5 or 10 nmol of CTAP alone significantly decreased the Tb, and 1.25 nmol of nor-BNI blocked that effect, indicating that the CTAP-induced hypothermia was kappa-mediated. The findings strongly suggest that mu antagonists, in blocking the basal hyperthermia mediated by mu receptors, can unmask the endogenous kappa receptor-mediated hypothermia, and that there is a tonic balance between mu and kappa opioid receptors that serves as a homeostatic mechanism for maintaining Tb.

  13. Requirement for caspase-8 in NF-kappaB activation by antigen receptor.

    Science.gov (United States)

    Su, Helen; Bidère, Nicolas; Zheng, Lixin; Cubre, Alan; Sakai, Keiko; Dale, Janet; Salmena, Leonardo; Hakem, Razqallah; Straus, Stephen; Lenardo, Michael

    2005-03-04

    Caspase-8, a proapoptotic protease, has an essential role in lymphocyte activation and protective immunity. We show that caspase-8 deficiency (CED) in humans and mice specifically abolishes activation of the transcription factor nuclear factor kappaB (NF-kappaB) after stimulation through antigen receptors, Fc receptors, or Toll-like receptor 4 in T, B, and natural killer cells. Caspase-8 also causes the alphabeta complex of the inhibitor of NF-kappaB kinase (IKK) to associate with the upstream Bcl10-MALT1 (mucosa-associated lymphatic tissue) adapter complex. Recruitment of the IKKalpha, beta complex, its activation, and the nuclear translocation of NF-kappaB require enzyme activity of full-length caspase-8. These findings thus explain the paradoxical association of defective apoptosis and combined immunodeficiency in human CED.

  14. Structure of the human [kappa]-opioid receptor in complex with JDTic

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Huixian; Wacker, Daniel; Mileni, Mauro; Katritch, Vsevolod; Han, Gye Won; Vardy, Eyal; Liu, Wei; Thompson, Aaron A.; Huang, Xi-Ping; Carroll, F. Ivy; Mascarella, S. Wayne; Westkaemper, Richard B.; Mosier, Philip D.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C. (VCU); (Scripps); (UNC); (Res. Tri. Inst.)

    2013-04-25

    Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and - in the case of {kappa}-opioid receptor ({kappa}-OR) - dysphoria and psychotomimesis. Here we report the crystal structure of the human {kappa}-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 {angstrom} resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human {kappa}-OR. Modelling of other important {kappa}-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for {kappa}-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human {kappa}-OR.

  15. Optimisation of in silico derived 2-aminobenzimidazole hits as unprecedented selective kappa opioid receptor agonists

    DEFF Research Database (Denmark)

    Sasmal, Pradip K; Krishna, C Vamsee; Sudheerkumar Adabala, S

    2015-01-01

    Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR...

  16. DMPD: Signaling to NF-kappaB by Toll-like receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18029230 Signaling to NF-kappaB by Toll-like receptors. Kawai T, Akira S. Trends Mo...l Med. 2007 Nov;13(11):460-9. Epub 2007 Oct 29. (.png) (.svg) (.html) (.csml) Show Signaling to NF-kappaB by... Toll-like receptors. PubmedID 18029230 Title Signaling to NF-kappaB by Toll-like receptors. Authors Kawai T

  17. Stereoselective synthesis and structure-affinity relationships of bicyclic kappa receptor agonists.

    Science.gov (United States)

    Kracht, Daniel; Rack, Elisabeth; Schepmann, Dirk; Fröhlich, Roland; Wünsch, Bernhard

    2010-01-07

    Reductive amination of the bicyclic ketone 4 led diastereoselectively to endo-configured amines, which were transformed into the amides 7-10. The synthesis of the diastereomers 25 with an exo-configured amino moiety at position 6 was only successful after deactivation of both N-atoms of the 1,4-diazabicyclo[3.3.1]nonane system. The N-1-oxide 19 with an N-4-tosyl moiety was the crucial intermediate, which allows SN2 substitution with NaN3 under inversion of the configuration at position 6. Whereas the endo-configured pyrrolidine 7a (WMS-1302) revealed a kappa receptor affinity of 73 nM, the exo-configured diastereomer 25a was almost inactive at the kappa receptor (Ki > 1 microM). Replacement of the 3,4-dichlorophenylacetyl residue by other acyl and sulfonyl residues showed that it is essential for high kappa affinity. The kappa receptor affinities of the conformationally constrained pyrrolidines 7a and 25a were correlated with the dihedral angle N(pyrrolidine)-C-C-N(acetamide). A systematic conformational analysis of the potent but flexible kappa agonist 2 showed that a dihedral angle of 168 degrees (as in 25a) is energetically more disfavored than a dihedral angle of 58 degrees (7a). However, even the conformation with a dihedral angle of 58 degrees does not represent an energy minimum, which might explain the reduced kappa affinity of 7a.

  18. Effects of kappa-opioid receptor ligands on intracranial self-stimulation in rats.

    Science.gov (United States)

    Todtenkopf, Mark S; Marcus, Jacqueline F; Portoghese, Philip S; Carlezon, William A

    2004-04-01

    Elevations in cAMP response element binding protein (CREB) function within the mesolimbic system of rats reduce cocaine reward in place conditioning studies and increase immobility in the forced swim test. Each of these behavioral adaptations can be interpreted as a depressive-like effect (i.e., anhedonia, despair) that may reflect reduced activity of brain reward systems. Furthermore, each effect appears due to increases in CREB-mediated expression of dynorphin, since each is attenuated by intracranial injections of the kappa-opioid receptor antagonist norBNI. Intracranial self-stimulation (ICSS) studies were conducted in rats to determine whether administration of a kappa-agonist would have depressive-like effects on brain stimulation reward, and whether pretreatment with a kappa-antagonist would attenuate any such effects. Conditions that have depressive effects in people (e.g., drug withdrawal) increase the threshold amounts of stimulation required to sustain ICSS in rats. Sprague-Dawley rats with lateral hypothalamic stimulating electrodes were tested in a "curve-shift" variant of the ICSS procedure after systemic administration of the kappa-agonist U-69593 alone, the novel kappa-antagonist 5'-acetamidinoethylnaltrindole (ANTI) alone, or co-administration of both drugs. U-69593 dose dependently increased ICSS thresholds, suggesting that activation of kappa-receptors reduced the rewarding impact of the brain stimulation. ANTI had no effects on its own, but it attenuated increases in ICSS thresholds caused by the agonist. These data provide further evidence that stimulation of brain kappa-receptors may trigger certain depressive-like signs, and that kappa antagonists may have efficacy as antidepressants without having reward-related actions of their own.

  19. Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display

    DEFF Research Database (Denmark)

    Gunnarsen, Kristin S; Kristinsson, Solveig G; Justesen, Sune

    2013-01-01

    We here report a novel phage display selection strategy enabling fast and easy selection of thermostabilized proteins. The approach is illustrated with stabilization of an aggregation-prone soluble single chain T cell receptor (scTCR) characteristic of the murine MOPC315 myeloma model. Random...

  20. Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis

    Directory of Open Access Journals (Sweden)

    Xuewei ePan

    2016-03-01

    Full Text Available Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The LPS profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor.

  1. Studies Toward the Pharmacophore of Salvinorin A, a Potent Kappa Opioid Receptor Agonist

    OpenAIRE

    Munro, Thomas A.; Mark A. Rizzacasa; Roth, Bryan L.; Toth, Beth A.; Yan, Feng

    2005-01-01

    Salvinorin A (1), from the sage Salvia divinorum, is a potent and selective kappa opioid receptor (KOR) agonist. We screened other salvinorins and derivatives for binding affinity and functional activity at opioid receptors. Our results suggest that the methyl ester and furan ring are required for activity, but that the lactone and ketone functionalities are not. Other salvinorins showed negligible binding affinity at the KOR. None of the compounds bound to mu or delta opioid receptors.

  2. Studies toward the pharmacophore of salvinorin A, a potent kappa opioid receptor agonist.

    Science.gov (United States)

    Munro, Thomas A; Rizzacasa, Mark A; Roth, Bryan L; Toth, Beth A; Yan, Feng

    2005-01-27

    Salvinorin A (1), from the sage Salvia divinorum, is a potent and selective kappa opioid receptor (KOR) agonist. We screened other salvinorins and derivatives for binding affinity and functional activity at opioid receptors. Our results suggest that the methyl ester and furan ring are required for activity but that the lactone and ketone functionalities are not. Other salvinorins showed negligible binding affinity at the KOR. None of the compounds bound to mu or delta opioid receptors.

  3. Changes of mu and kappa opioid receptors in cathartic colon of rat

    Institute of Scientific and Technical Information of China (English)

    LIU Bao-hua; MO Ping; JIA Hou-jun; LI Chun-xue; ZHANG Sheng-ben

    2004-01-01

    Objective: To oberve the changes of mu and kappa opioid receptors in the cathartic colon of rat, and to clarify that whether opioid receptors accounts for the occurrence of slow trait constipation (STC). Methods: The cathartiic colon model of rat was made by feeding with laxatives. The activity of mu and kappa opioid receptors in the cathartic colon of rat was measured by radio-ligand binding assay. Results: Compared with the control group, the maximal binding capacity (Bmax) and affinity(Kd) of mu opioid receptor in cathartic colon group were significantly increased (207. 00 ± 22. 90 fmol/mg·p vs 82. 00 ± 14.23 fmol/mg· p, P < 0.01 ;3.30 ± 0.45 mmol/L vs 2.40 ± 0.57 mmol/L, P < 0.05). The maximal binding capacity of kappa opioid receptor also showed a great increase (957. 00 ± 102. 41 fmol/mg· p vs 459.00 ± 52.41 fmol/mg·p, P < 0.01 ), but no significant difference of affinity was found between the two groups. Conclsion: The mu and kappa opioid receptors may be involved in the functional disorders of cathartic colon.

  4. Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

    DEFF Research Database (Denmark)

    Mahony, Jennifer; Kot, Witold Piotr; Murphy, James;

    2013-01-01

    Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been impli...

  5. Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

    DEFF Research Database (Denmark)

    Mahony, Jennifer; Kot, Witold Piotr; Murphy, James

    2013-01-01

    Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been impli...

  6. A novel phage-library-selected peptide inhibits human TNF-α binding to its receptors.

    Science.gov (United States)

    Brunetti, Jlenia; Lelli, Barbara; Scali, Silvia; Falciani, Chiara; Bracci, Luisa; Pini, Alessandro

    2014-06-03

    We report the identification of a new human tumor necrosis factor-alpha (TNF-α) specific peptide selected by competitive panning of a phage library. Competitive elution of phages was obtained using the monoclonal antibody adalimumab, which neutralizes pro-inflammatory processes caused by over-production of TNF-α in vivo, and is used to treat severe symptoms of rheumatoid arthritis. The selected peptide was synthesized in monomeric and branched form and analyzed for binding to TNF-α and competition with adalimumab and TNF-α receptors. Results of competition with TNF-α receptors in surface plasmon resonance and melanoma cells expressing both TNF receptors make the peptide a candidate compound for the development of a novel anti-TNF-α drug.

  7. NF-kappaB signaling mediates vascular smooth muscle endothelin type B receptor expression in resistance arteries

    DEFF Research Database (Denmark)

    Zheng, Jian-Pu; Zhang, Yaping; Edvinsson, Lars

    2010-01-01

    Vascular smooth muscle cells (SMC) endothelin type B (ET(B)) receptor upregulation results in strong vasoconstriction and reduction of local blood flow. We hypothesizes that the underlying molecular mechanisms involve transcriptional factor nuclear factor-kappaB (NF-kappaB) pathway. ET(B) receptor...

  8. NF-kappaB activity marks cells engaged in receptor editing.

    Science.gov (United States)

    Cadera, Emily J; Wan, Fengyi; Amin, Rupesh H; Nolla, Hector; Lenardo, Michael J; Schlissel, Mark S

    2009-08-03

    Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

  9. Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

    NARCIS (Netherlands)

    Kuznetsova, T.; Wang, S.Y.; Rao, N.A.; Mandoli, A.; Martens, J.H.; Rother, N; Aartse, A.; Groh, L.; Janssen-Megens, E.M.; Li, G.; Ruan, Y.; Logie, C.; Stunnenberg, H.G.

    2015-01-01

    BACKGROUND: The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activ

  10. Kappa Opioid Receptors Mediate where Fear Is Expressed Following Extinction Training

    Science.gov (United States)

    Cole, Sindy; Richardson, Rick; McNally, Gavan P.

    2011-01-01

    Six experiments used a within-subjects renewal design to examine the involvement of kappa opioid receptors (KORs) in regulating the expression and recovery of extinguished fear. Rats were trained to fear a tone conditioned stimulus (CS) via pairings with foot shock in a distinctive context (A). This was followed by extinction training of the CS in…

  11. Kappa Opioid Receptors Mediate where Fear Is Expressed Following Extinction Training

    Science.gov (United States)

    Cole, Sindy; Richardson, Rick; McNally, Gavan P.

    2011-01-01

    Six experiments used a within-subjects renewal design to examine the involvement of kappa opioid receptors (KORs) in regulating the expression and recovery of extinguished fear. Rats were trained to fear a tone conditioned stimulus (CS) via pairings with foot shock in a distinctive context (A). This was followed by extinction training of the CS in…

  12. Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

    NARCIS (Netherlands)

    Kuznetsova, T.; Wang, S.Y.; Rao, N.A.; Mandoli, A.; Martens, J.H.; Rother, N; Aartse, A.; Groh, L.; Janssen-Megens, E.M.; Li, G.; Ruan, Y.; Logie, C.; Stunnenberg, H.G.

    2015-01-01

    BACKGROUND: The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by

  13. The mu1, mu2, delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine

    DEFF Research Database (Denmark)

    Kristensen, K; Christensen, C B; Christrup, Lona Louring

    1995-01-01

    The binding affinities of racemic methadone and its optical isomers R-methadone and S-methadone were evaluated for the opioid receptors mu1, mu2, delta and kappa, in comparison with that of morphine. The analgesic R-methadone had a 10-fold higher affinity for mu1 receptors than S-methadone (IC50 3.......0 nM and 26.4 nM, respectively). At the mu2 receptor, the IC50 value of R-methadone was 6.9 nM and 88 nM for S-methadone, respectively. As expected, R-methadone had twice the affinity for mu1 and mu2 receptors than the racemate. All of the compounds tested had low affinity for the delta and kappa...

  14. Avance en el diseño de un péptido bloqueador del receptor Opioide Kappa 2 humano

    Directory of Open Access Journals (Sweden)

    Velásquez Álvarez Alvaro Andrés

    2000-12-01

    Full Text Available

    Se evaluó la posibilidad de predecir una probable estructura secundaria para el Receptor Opioide Kappa 2 humano tomando como base la secuencia de aminoácidos del Receptor Opioide Kappa 1 humano. La estructura predicha mostró ser compatible con los datos que se poseen acerca de este tipo de receptores. Con esta prueba inicial, el proyecto que tiene como objetivo principal diseñar un análogo proteico para el Receptor Opioide Kappa 2 humano, ha mostrado el nivel mínimo de viabilidad necesario para ser continuado.

  15. AVANCE lEN IEL DISEÑO DIE UN PÉPTIDO BLOQUEADOR DIEL RECEPTOR OPIOIDIE KAPPA 2 HUMANO

    Directory of Open Access Journals (Sweden)

    Sonia Viviana Beltrán Velásquez

    2000-12-01

    Full Text Available Se evaluó la posibilidad de predecir una probable estructura secundaria para el Receptor Opioide Kappa 2 humano tomando como base la secuencia de aminoácidos del Receptor Opioide Kappa 1 humano. La estructura predicha mostró ser compatible con los datos que se poseen acerca de este tipo de receptores. Con esta prueba inicial, el proyecto que tiene como objetivo principal el diseñar un análogo proteico para el ReceptorOpioide Kappa 2 humano, ha mostrado el nivel mínimo de viabilidad necesario para ser continuado.

  16. D3 dopamine and kappa opioid receptor alterations in human brain of cocaine-overdose victims.

    Science.gov (United States)

    Mash, D C; Staley, J K

    1999-06-29

    Cocaine is thought to be addictive because chronic use leads to molecular adaptations within the mesolimbic dopamine (DA) circuitry, which affects motivated behavior and emotion. Although the reinforcing effects of cocaine are mediated primarily by blockade of DA uptake, reciprocal signaling between DA and endogenous opioids has important implications for understanding cocaine dependence. We have used in vitro autoradiography and ligand binding to map D3 DA and kappa opioid receptors in the human brains of cocaine-overdose victims. The number of D3 binding sites was increased one-to threefold over the nucleus accumbens and ventromedial sectors of the caudate and putamen from cocaine-overdose victims, as compared to age-matched and drug-free control subjects. D3 receptor/cyclophilin mRNA ratios in the nucleus accumbens were increased sixfold in cocaine-overdose victims over control values, suggesting that cocaine exposure also affects the expression of D3 receptor mRNA. The number of kappa opioid receptors in the nucleus accumbens and other corticolimbic areas from cocaine fatalities was increased twofold as compared to control values. Cocaine-overdose victims exhibiting preterminal excited delirium had a selective upregulation of kappa receptors measured also in the amygdala. Understanding the complex regulatory profiles of DA and opioid synaptic markers that occur with chronic misuse of cocaine may suggest multitarget strategies for treating cocaine dependence.

  17. Kappa opioid receptor antagonist and N-methyl-D- aspartate receptor antagonist affect dynorphin- induced spinal cord electrophysiologic impairment

    Institute of Scientific and Technical Information of China (English)

    Yu Chen; Liangbi Xiang; Jun Liu; Dapeng Zhou; Hailong Yu; Qi Wang; Wenfeng Han; Weijian Ren

    2012-01-01

    The latencies of motor- and somatosensory-evoked potentials were prolonged to different degrees, and wave amplitude was obviously decreased, after injection of dynorphin into the rat subarachnoid cavity.The wave amplitude and latencies of motor- and somatosensory-evoked potentials were significantly recovered at 7 and 14 days after combined injection of dynorphin and either the kappa opioid receptor antagonist nor-binaltorphimine or the N-methyl-D-aspartate receptor antagonist MK-801.The wave amplitude and latency were similar in rats after combined injection of dynorphin and nor-binaltorphimine or MK-801.These results suggest that intrathecal injection of dynorphin causes damage to spinal cord function.Prevention of N-methyl-D-aspartate receptor or kappa receptor activation lessened the injury to spinal cord function induced by dynorphin.

  18. Effects of kappa opioid receptor-selective agonists on responses of pelvic nerve afferents to noxious colorectal distension.

    Science.gov (United States)

    Su, X; Sengupta, J N; Gebhart, G F

    1997-08-01

    The aim of this study was to examine the effects of kappa-opioid receptor selective agonists on responses of mechanosensitive afferent fibers in the pelvic nerve. Single-fiber recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root of the rat. A total of 572 afferent fibers in the S1 dorsal root were identified by electrical stimulation of the pelvic nerve; 252 (44%) responded to noxious colorectal distension (CRD; 80 mmHg). Of these 252 fibers that responded to CRD, 100 were studied further. All 100 fibers gave monotonic increases in firing to increasing pressures of CRD. Eighty-eight fibers had low thresholds for response (mean: 3 mmHg) and 12 fibers had high-thresholds for response (mean: 28 mmHg). Responses of 17 fibers also were tested after instillation of 5% mustard oil (MO) into the colon. The resting activity of 16/17 fibers significantly increased after MO instillation; 13 (77%) also exhibited sensitization of responses to graded CRD when tested 30 min after intracolonic MO instillation. The effects of kappa1-opioid receptor preferring agonists (U50,488H, U69,593 and U62,066), the kappa2-opioid receptor preferring agonist bremazocine, and the kappa3-opioid receptor preferring agonist naloxone benzoylhydrazone (nalBzoH) were tested on responses of 64 mechanosensitive afferent fibers to noxious CRD. All five agonists dose-dependently inhibited afferent fiber responses to noxious CRD. Doses producing inhibition to 50% of the control response to CRD did not differ among the five agonists, ranging from approximately 4 to 15 mg/kg. The effects of kappa1, kappa2, and kappa3 receptor agonists were attenuated by naloxone; two kappa-opioid receptor-selective antagonists were ineffective. There were no differences in the dose-response relationships of these drugs for fibers recorded from untreated and irritant-treated colons. Conduction velocities of the fibers remained unaffected after high doses of all tested agonists. In an in vitro

  19. Peptide Phage Display as a Tool for Drug Discovery: Targeting Membrane Receptors

    Directory of Open Access Journals (Sweden)

    Tomaz Bratkovic

    2011-01-01

    Full Text Available Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein’s activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities. Because isolating or producing recombinant membrane proteins for use as target molecules in library screening is often impossible, innovative selection strategies such as panning against whole cells or tissues, recombinant receptor ectodomains, or neutralizing antibodies to endogenous binding partners were devised. Prominent examples from a two-decade history of peptide phage display will be presented, focusing on the design of affinity selection experiments, methods for improving the initial hits, and applications of the identified peptides.

  20. Synthesis of [{sup 3}]DIPPA: a potent irreversible antagonist selective for the {kappa} opioid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Anchih; Portoghese, P.S. [Minnesota Univ., Minneapolis, MN (United States). College of Pharmacy; Trometer, J.D. [E.I. Du Pont de Nemours and Co., Inc., Boston, MA (United States)

    1995-06-01

    2-(3,4-Dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophe nyl)-2-(1-pyrrolidinyl)ethyl]acetamide (1,DIPPA) has been previously reported to be an opioid receptor affinity label that produces selective and long-lasting {kappa} opioid receptor antagonism in mice. High specific activity [{sup 3}H]DIPPA (39.7 Ci/mmol) was prepared by bromination and catalytic tritiation of the amino precursor of DIPPA followed by conversion to the isothiocyanate with thiophosgene. (Author).

  1. A Genotypic Analysis of Five P. aeruginosa Strains after Biofilm Infection by Phages Targeting Different Cell Surface Receptors

    Directory of Open Access Journals (Sweden)

    Diana P. Pires

    2017-06-01

    Full Text Available Antibiotic resistance constitutes one of the most serious threats to the global public health and urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as being good alternatives to traditional antibiotic therapies. In this study, the efficacy of phages, targeting different cell receptors, against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48 h. Although significant reductions in the number of viable cells were achieved for both cases, the high level of adaptability of the bacteria in response to the selective pressure caused by phage treatment resulted in the emergence of phage-resistant variants. To further investigate the genetic makeup of phage-resistant variants isolated from biofilm infection experiments, some of these bacteria were selected for phenotypic and genotypic characterization. Whole genome sequencing was performed on five phage-resistant variants and all of them carried mutations affecting the galU gene as well as one of pil genes. The sequencing analysis further revealed that three of the P. aeruginosa PAO1 variants carry large deletions (>200 kbp in their genomes. Complementation of the galU mutants with wild-type galU in trans restored LPS expression on the bacterial cell surface of these bacterial strains and rendered the complemented strains to be sensitive to phages. This provides unequivocal evidence that inactivation of galU function was associated with resistance to the phages that uses LPS as primary receptors. Overall, this work demonstrates that P. aeruginosa biofilms can survive phage attack and develop phage-resistant variants exhibiting defective LPS production and loss of type IV pili that are well adapted to the biofilm mode of growth.

  2. [{sup 11}C]-MeJDTic: a novel radioligand for {kappa}-opioid receptor positron emission tomography imaging

    Energy Technology Data Exchange (ETDEWEB)

    Poisnel, Geraldine; Oueslati, Farhana; Dhilly, Martine; Delamare, Jerome [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France); Perrio, Cecile [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: perrio@cyceron.fr; Debruyne, Daniele [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: debruyne@cyceron.fr; Barre, Louisa [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)

    2008-07-15

    Introduction: Radiopharmaceuticals that can bind selectively the {kappa}-opioid receptor may present opportunities for staging clinical brain disorders and evaluating the efficiency of new therapies related to stroke, neurodegenerative diseases or opiate addiction. The N-methylated derivative of JDTic (named MeJDTic), which has been recently described as a potent and selective antagonist of {kappa}-opioid receptor in vitro, was labeled with carbon-11 and evaluated for in vivo imaging the {kappa}-opioid receptor in mice. Methods: [{sup 11}C]-MeJDTic was prepared by methylation of JDTic with [{sup 11}C]-methyl triflate. The binding of [{sup 11}C]-MeJDTic to {kappa}-opioid receptor was investigated ex vivo by biodistribution and competition studies using nonfasted male CD1 mice. Results: [{sup 11}C]-MeJDTic exhibited a high and rapid distribution in peripheral organs. The uptake was maximal in lung where the {kappa} receptor is largely expressed. [{sup 11}C]-MeJDTic rapidly crossed the blood-brain barrier and accumulated in the brain regions of interest (hypothalamus). The parent ligand remained the major radioactive compound in brain during the experiment. Chase studies with U50,488 (a {kappa} referring agonist), morphine (a {mu} agonist) and naltrindole (a {delta} antagonist) demonstrated that this uptake was the result of specific binding to the {kappa}-opioid receptor. Conclusion: These findings suggested that [{sup 11}C]-MeJDTic appeared to be a promising selective 'lead' radioligand for {kappa}-opioid receptor PET imaging.

  3. NF-kappaB signaling mediates vascular smooth muscle endothelin type B receptor expression in resistance arteries

    DEFF Research Database (Denmark)

    Zheng, Jian-Pu; Zhang, Yaping; Edvinsson, Lars

    2010-01-01

    Vascular smooth muscle cells (SMC) endothelin type B (ET(B)) receptor upregulation results in strong vasoconstriction and reduction of local blood flow. We hypothesizes that the underlying molecular mechanisms involve transcriptional factor nuclear factor-kappaB (NF-kappaB) pathway. ET(B) receptor...... upregulation and activation of NF-kappaB were studied at functional contraction (in vitro myograph), mRNA (real-time PCR), and protein (Western blot and immunocytochemistry) levels during organ culture of rat mesenteric arteries. Organ culture of the artery segments induced a time-dependent strong contractile...... response to sarafotoxin 6c in parallel with enhanced expression of ET(B) receptor mRNA and protein in the SMC. Western blot experiments demonstrated that phosphorylation of NF-kappaB p65 was time-dependently induced during organ culture starting at 1h. In addition, cytoplasmic IkB degradation occurred...

  4. Complete knockout of the nociceptin/orphanin FQ receptor in the rat does not induce compensatory changes in mu, delta and kappa opioid receptors.

    NARCIS (Netherlands)

    Homberg, J.R.; Mul, J.D.; Wit, E. de; Cuppen, E.

    2009-01-01

    The nociceptin/orphanin FQ (N/OFQ) opioid peptide receptor (NOPr) is a new member of the opioid receptor family consisting of mu, delta and kappa opioid receptors. The anti-opioid properties of its endogenous ligand, N/OFQ provide the receptor interesting potentials in symptoms and processes related

  5. Complete knockout of the nociceptin/orphanin FQ receptor in the rat does not induce compensatory changes in mu, delta and kappa opioid receptors.

    NARCIS (Netherlands)

    Homberg, J.R.; Mul, J.D.; de Wit, E.; Cuppen, E.

    2009-01-01

    The nociceptin/orphanin FQ (N/OFQ) opioid peptide receptor (NOPr) is a new member of the opioid receptor family consisting of mu, delta and kappa opioid receptors. The anti-opioid properties of its endogenous ligand, N/OFQ provide the receptor interesting potentials in symptoms and processes related

  6. Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5.

    Science.gov (United States)

    Li, Chuanzhao; Plugariu, Carmela G; Bajgier, Joanna; White, John R; Liefer, Kristin M; Wu, Sheng-Jiun; Chaiken, Irwin

    2002-01-01

    Phage display was used to identify sequences that mimic structural determinants in interleukin5 (IL5) for IL5 receptor recognition. A coiled coil stem loop (CCSL) miniprotein scaffold library was constructed with its turn region randomized and panned for binding variants against human IL5 receptor alpha chain (IL5Ralpha). Competition enzyme-linked immunosorbent assays identified CCSL-phage selectants for which binding to IL5Ralpha was competed by IL5. The most frequently selected and IL5-competed CCSL-phage contain charged residues Arg and Glu in their turn sequences, in this regard resembling a beta strand sequence in the 'CD turn' region, of IL5, that has been proposed to present a key determinant for IL5 receptor alpha chain recognition. The most dominant CCSL-phage selectant sequence, PVEGRV, contains a negative/positive charge pattern similar to that seen in the original CD turn. To test the relatedness of CCSL-phage selectant sequences to the IL5 receptor recognition epitope, PVEGRV was grafted into the sequence 87--92 of a monomeric IL5. The resulting IL5 variant, [(87)PVEGRV(92)]GM1, was able to bind to IL5Ralpha in biosensor assays, to elicit TF-1 cell proliferation and to induce STAT5 phosphorylation in TF-1 cells. The results help discern sequence patterns in the IL5 CD turn region which are key in driving receptor recognition and demonstrate the utility of CCSL miniprotein scaffold phage display to identify local IL5 mimetic sequence arrangements that may ultimately lead to IL5 antagonists.

  7. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    DEFF Research Database (Denmark)

    Shen, P; Canoll, P D; Sap, J

    1999-01-01

    Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions...... in vitro [Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, J. Sap, Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region, Mol. Cell. Biol. 13...... processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene...

  8. Locus coeruleus kappa-opioid receptors modulate reinstatement of cocaine place preference through a noradrenergic mechanism.

    Science.gov (United States)

    Al-Hasani, Ream; McCall, Jordan G; Foshage, Audra M; Bruchas, Michael R

    2013-11-01

    Activation of kappa-opioid receptors (KORs) in monoamine circuits results in dysphoria-like behaviors and stress-induced reinstatement of drug seeking in both conditioned place preference (CPP) and self-administration models. Noradrenergic (NA) receptor systems have also been implicated in similar behaviors. Dynorphinergic projections terminate within the locus coeruleus (LC), a primary source of norepinephrine in the forebrain, suggesting a possible link between the NA and dynorphin/kappa opioid systems, yet the implications of these putative interactions have not been investigated. We isolated the necessity of KORs in the LC in kappa opioid agonist (U50,488)-induced reinstatement of cocaine CPP by blocking KORs in the LC with NorBNI (KOR antagonist). KOR-induced reinstatement was significantly attenuated in mice injected with NorBNI in the LC. To determine the sufficiency of KORs in the LC on U50,488-induced reinstatement of cocaine CPP, we virally re-expressed KORs in the LC of KOR knockout mice. We found that KORs expression in the LC alone was sufficient to partially rescue KOR-induced reinstatement. Next we assessed the role of NA signaling in KOR-induced reinstatement of cocaine CPP in the presence and absence of a α2-agonist (clonidine), β-adrenergic receptor antagonist (propranolol), and β(1)- and β(2)-antagonist (betaxolol and ICI-118,551 HCl). Both the blockade of postsynaptic β(1)-adrenergic receptors and the activation of presynaptic inhibitory adrenergic autoreceptors selectively potentiated the magnitude of KOR-induced reinstatement of cocaine CPP but not cocaine-primed CPP reinstatement. Finally, viral restoration of KORs in the LC together with β-adrenergic receptor blockade did not potentiate KOR-induced reinstatement to cocaine CPP, suggesting that adrenergic receptor interactions occur at KOR-expressing regions external to the LC. These results identify a previously unknown interaction between KORs and NA systems and suggest a NA

  9. Effect of GNTI,a kappa opioid receptor antagonist, on MK-801-induced hyperlocomotion and stereotypy in mice

    Institute of Scientific and Technical Information of China (English)

    Chun-ting QI; Hong ZOU; Chen-hao ZHANG; Qing-lian XIE; Mei-lei JIN; Lei YU

    2006-01-01

    Aim:To examine the effect of GNTI[5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,-5α-epoxy-3-14-dihydroxy-6,7-2',3'-indolomorphinan],a selective antagonist for the kappa opioid receptor,in the MK-801 (dizocilpine maleate)-induced behavioral model of psychosis in schizophrenia as a way to explore the involvement of the kappa opioid receptor in modulating psychotic symptoms of schizophrenia.Methods:Two doses 0f MK-801 (0.3 mg/kg and 0.6 mg/kg) were administered by systemic injection in mice to induce psychosis-like behavior as a rodent schizophrenia model, preceded by an injection of different doses of GNTI. Both locomotion and stereotypy were measured as the behavioral endpoints for quantitative analysis.Results:GNTI inhibited MK-801-induced hyperlocomotion and stereotypy.In particular,GNTI showed differential modulation of stereotypy induced by 0.3 mg/kg VS 0.6 mg/kg MK-801.Conclusion:Antagonism of kappa opioid receptors attenuates MK-801-induced behavior,suggesting a potential involvement of the kappa opioid receptor in psychosis-like symptoms of schizophrenia.GNTI aDpears to be a useful pharmacological tool to explore the kappa opioid receptor function in vivo.

  10. Localization of the kappa opioid receptor gene to human chromosome band 8q11. 2

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kazuki; Takeda, Jun; Bell, G.I.; Espinosa, R.; Le Beau, M.M. (Univ. of Chicago, IL (United States))

    1994-02-01

    Using the cloned mouse kappa opioid receptor cDNA clone as a probe, screened a human genomic library and isolated a clone containing part of the human kappa opioid receptor gene (OPRK1), designated [lambda]hSR4-1. To determine the chromosomal localization of OPRK1, [lambda]hSR4-1 DNA was labeled with biotin by nick-translation in the presence of bio-11-dUTP and hybridized to human metaphase cells prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes as described previously. Hybridization of the OPRK1-specific probe [lambda]hSR4-1 DNA to normal human metaphase chromosomes resulted in specific labeling only of chromosome 8. Specific labeling of 8q11 was observed on all 4 (6 cells), 3 (9 cells), 2 (9 cells), or 1 (1 cell) chromatid of the chromosome 8 homologs in 25 cells examined. Of 72 signals observed, 70 were located at 8q11. 1 signal was located at 7q11 and at 12p11. In most cells, the signal on 8q was located at 8q11.2. 7 refs., 1 fig.

  11. Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

    DEFF Research Database (Denmark)

    Sørensen, Martine C. Holst; Gencay, Yilmaz Emre; Birk, Tina;

    2015-01-01

    were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according......In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated...... therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages...

  12. Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

    Science.gov (United States)

    Sirohi, Sunil; Walker, Brendan M

    2015-11-01

    Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

  13. Effects of salvinorin A, a kappa-opioid hallucinogen, on a neuroendocrine biomarker assay in nonhuman primates with high kappa-receptor homology to humans.

    Science.gov (United States)

    Butelman, Eduardo R; Mandau, Marek; Tidgewell, Kevin; Prisinzano, Thomas E; Yuferov, Vadim; Kreek, Mary Jeanne

    2007-01-01

    This study focused on the in vivo effects of the kappa-opioid hallucinogen salvinorin A, derived from the plant Salvia divinorum. The effects of salvinorin A (0.0032-0.056 mg/kg i.v.) were studied in a neuroendocrine biomarker assay of the anterior pituitary hormone prolactin in gonadally intact, adult male and female rhesus monkeys (n = 4 each). Salvinorin A produced dose- and time-dependent neuroendocrine effects, similar to the synthetic high-efficacy kappa-agonist U69,593 ((+)-(5alpha,7 alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiniyl)-1-oxaspiro[4.5]dec-8yl]-benzeneacetamide), but of shorter duration than the latter. Salvinorin A was approximately equipotent to U69,593 in this endpoint (salvinorin A ED50, 0.015 mg/kg; U69,593 ED(50), 0.0098 mg/kg). The effects of i.v. salvinorin A were not prevented by a small dose of the opioid antagonist nalmefene (0.01 mg/kg s.c.) but were prevented by a larger dose of nalmefene (0.1 mg/kg); the latter nalmefene dose is sufficient to produce kappa-antagonist effects in this species. In contrast, the 5HT2 receptor antagonist ketanserin (0.1 mg/kg i.m.) did not prevent the effects of salvinorin A. As expected, the neuroendocrine effects of salvinorin A (0.0032 mg/kg i.v.) were more robust in female than in male subjects. Related studies focused on full-length cloning of the coding region of the rhesus monkey kappa-opioid receptor (OPRK1) gene and revealed a high homology of the nonhuman primate OPRK1 gene compared with the human OPRK1 gene, including particular C-terminal residues thought to be involved in receptor desensitization and internalization. The present studies indicate that the hallucinogen salvinorin A acts as a high-efficacy kappa-agonist in nonhuman primates in a translationally viable neuroendocrine biomarker assay.

  14. Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

    DEFF Research Database (Denmark)

    Sørensen, Martine C. Holst; Gencay, Yilmaz Emre; Birk, Tina;

    2015-01-01

    In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated...

  15. Use of phage display to identify novel mineralocorticoid receptor-interacting proteins.

    Science.gov (United States)

    Yang, Jun; Fuller, Peter J; Morgan, James; Shibata, Hirotaka; McDonnell, Donald P; Clyne, Colin D; Young, Morag J

    2014-09-01

    The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter.

  16. A unique binding epitope for salvinorin A, a non-nitrogenous kappa opioid receptor agonist.

    Science.gov (United States)

    Kane, Brian E; Nieto, Marcelo J; McCurdy, Christopher R; Ferguson, David M

    2006-05-01

    Salvinorin A is a potent kappa opioid receptor (KOP) agonist with unique structural and pharmacological properties. This non-nitrogenous ligand lacks nearly all the structural features commonly associated with opioid ligand binding and selectivity. This study explores the structural basis to salvinorin A binding and selectivity using a combination of chimeric and single-point mutant opioid receptors. The experiments were designed based on previous models of salvinorin A that locate the ligand within a pocket formed by transmembrane (TM) II, VI, and VII. More traditional sites of opioid recognition were also explored, including the highly conserved aspartate in TM III (D138) and the KOP selectivity site E297, to determine the role, if any, that these residues play in binding and selectivity. The results indicate that salvinorin A recognizes a cluster of residues in TM II and VII, including Q115, Y119, Y312, Y313, and Y320. Based on the position of these residues within the receptor, and prior study on salvinorin A, a model is proposed that aligns the ligand vertically, between TM II and VII. In this orientation, the ligand spans residues that are spaced one to two turns down the face of the helices within the receptor cavity. The ligand is also in close proximity to EL-2 which, based on chimeric data, is proposed to play an indirect role in salvinorin A binding and selectivity.

  17. Analgesia produced by exposure to 2450-MHz radiofrequency radiation (RFR) is mediated by brain mu- and kappa-opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Salomon, G.; Park, E.J.; Quock, R.M. (Univ. of Illinois, Rockford (United States))

    1992-02-26

    This study was conducted to identify the opioid receptor subtype(s) responsible for RFR-induced analgesia. Male Swiss Webster mice, 20-25 g, were exposed to 20 mW/cm{sup 2} RFR in a 2,450-MHz waveguide system for 10 min, then tested 15 min later in the abdominal constriction paradigm which detects {mu}- and {kappa}-opioid activity. Immediately following RFR exposure, different groups of mice were pretreated intracerebroventricularly with different opioid receptor blockers with selectivity for {mu}- or {kappa}-opioid receptors. Results show that RFR-induced analgesia was attenuated by higher but not lower doses of the non-selective antagonist naloxone, but the selective {mu}-opioid antagonist {beta}-funaltrexamine and by the selective {kappa}-opioid antagonist norbinaltorphimine. RFR-induced analgesia was also reduced by subcutaneous pretreatment with 5.0 mg/kg of the {mu}-/{kappa}-opioid antagonist({minus})-5,9-diethyl-{alpha}-5,9-dialkyl-2{prime}-hydroxy-6,7-benzomorphan(MR-2266). These findings suggest that RFR-induced analgesia may be mediated by both {mu}- and {kappa}-opioid mechanisms.

  18. Evaluation of the kappa-opioid receptor-selective tracer [{sup 11}C]GR103545 in awake rhesus macaques

    Energy Technology Data Exchange (ETDEWEB)

    Schoultz, Bent W. [University of Oslo, Department of Chemistry, Oslo (Norway); Hjornevik, Trine; Willoch, Frode [University of Oslo, Centre for Molecular Biology and Neuroscience and Institute of Basic Medical Sciences, Oslo (Norway); Akershus University Hospital, Department of Nuclear Medicine, Loerenskog (Norway); Marton, Janos [ABX Advanced Biochemical Compounds GmbH, Radeberg (Germany); Noda, Akihiro; Murakami, Yoshihiro; Miyoshi, Sosuke; Nishimura, Shintaro [Medical and Pharmacological Research Center Foundation, Basic Research Department, Hakui City, Ishikawa (Japan); Aarstad, Erik [University College of London, Institute of Nuclear Medicine, London (United Kingdom); Drzezga, Alexander [Technische Universitaet Muenchen, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany); Matsunari, Ichiro [Medical and Pharmacological Research Center Foundation, Clinical Research Department, Hakui City, Ishikawa (Japan); Henriksen, Gjermund [University of Oslo, Department of Chemistry, Oslo (Norway); Technische Universitaet Muenchen, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany)

    2010-06-15

    The recent development in radiosynthesis of the {sup 11}C-carbamate function increases the potential of [{sup 11}C]GR103545, which for the last decade has been regarded as promising for imaging the kappa-opioid receptor ({kappa}-OR) with PET. In the present study, [{sup 11}C]GR103545 was evaluated in awake rhesus macaques. Separate investigations were performed to clarify the OR subtype selectivity of this compound. Regional brain uptake kinetics of [{sup 11}C]GR103545 was studied 0-120 min after injection. The binding affinity and opioid subtype selectivity of [{sup 11}C]GR103545 was determined in cells transfected with cloned human opioid receptors. In vitro binding assays demonstrated a high affinity of GR103545 for {kappa}-OR (K{sub i} = 0.02 {+-}0.01 nM) with excellent selectivity over {mu}-OR (6 x 10{sup 2}-fold) and {delta}-OR (2 x 10{sup 4}-fold). PET imaging revealed a volume of distribution (V{sub T}) pattern consistent with the known distribution of {kappa}-OR, with striatum = temporal cortex > cingulate cortex > frontal cortex > parietal cortex > thalamus > cerebellum. [{sup 11}C]GR103545 is selective for {kappa}-OR and holds promise for use to selectively depict and quantify this receptor in humans by means of PET. (orig.)

  19. The crystal structure of a bimorphinan with highly selective kappa opioid receptor antagonist activity

    Science.gov (United States)

    Urbańczyk-Lipkowska, Zofia; Etter, Margaret C.; Lipkowski, Andrzej W.; Portoghese, Philip S.

    1987-07-01

    The crystal structure of the dihydrobromide heptahydrate of nor-binaltorphimine (17, 17'-bis(cyclopropylmethyl)-6,6',7,7'-tetrahydro-4,5α: 4',5'α-diepoxy-6,6'-imino[7,7' bimorphinan]-3,3',14,14'-tetraol)is presented. This structure is the first reported structure of a rigid bivalent opioid ligand. Two morphinan pharmacophores are connected by a rigid spacer, the pyrrole ring. The nor-binaltorphimine structure itself shows unique, high selectivity as a kappa opioid receptor antagonist. Crystal data: P3 2, Z = 3, a = b = 20.223 (4), c = 9.541(7) Å, α = β = 90°, γ = 120°; R = 0.079 (1765 reflections, Fobs > 1σ( F)).

  20. Antinociceptive and hypothermic effects of Salvinorin A are abolished in a novel strain of kappa-opioid receptor-1 knockout mice.

    Science.gov (United States)

    Ansonoff, Michael A; Zhang, Jiwen; Czyzyk, Traci; Rothman, Richard B; Stewart, Jeremy; Xu, Heng; Zjwiony, Jordan; Siebert, Daniel J; Yang, Feng; Roth, Bryan L; Pintar, John E

    2006-08-01

    Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the kappa-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as kappa-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 microg of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of kappa-opioid receptor knockout mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional kappa-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for kappa 1- but not kappa 2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for kappa(1)-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the kappa-opioid receptor.

  1. Toward a structure-based model of salvinorin A recognition of the kappa-opioid receptor.

    Science.gov (United States)

    Kane, Brian E; McCurdy, Christopher R; Ferguson, David M

    2008-03-27

    The structural basis to salvinorin A recognition of the kappa-opioid receptor is evaluated using a combination of site-directed mutagenesis and molecular-modeling techniques. The results show that salvinorin A recognizes a collection of residues in transmembrane II and VII, including Q115, Y119, Y313, I316, and Y320. The mutation of one hydrophobic residue in particular, I316, was found to completely abolish salvinorin A binding. As expected, none of the residues in transmembrane III or VI commonly associated with opiate recognition (such as D138 or E297) appear to be required for ligand binding. On the basis of the results presented here and elsewhere, a binding site model is proposed that aligns salvinorin A vertically within a pocket spanning transmembrane II and VII, with the 2' substituent directed toward the extracellular domains. The model explains the role that hydrophobic contacts play in binding this lipophilic ligand and gives insight into the structural basis to the mu-opioid receptor selectivity of 2'-benzoyl salvinorin (herkinorin).

  2. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

    Institute of Scientific and Technical Information of China (English)

    GENG De-chun; XU Yao-zeng; YANG Hui-lin; ZHU Guang-ming; WANG Xian-bin; ZHU Xue-song

    2011-01-01

    Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of >100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

  3. The kappa-opioid receptor is involved in the stimulating effect of nicotine on adrenocortical activity but not in nicotine induced anxiety.

    Science.gov (United States)

    Marco, Eva Maria; Llorente, Ricardo; Pérez-Alvarez, Laura; Moreno, Enrique; Guaza, Carmen; Viveros, Maria Paz

    2005-09-01

    The kappa (kappa) opioid system appears to interact with nicotine in the modulation of locomotion and addiction related processes. In this study we have investigated the possible implication of the kappa-opioid system in the effects of nicotine on anxiety and adrenocortical activity. In two different experiments, we analysed the possible interaction between nicotine (0.5 mg/kg i.p.) and either the kappa-opioid receptor antagonist nor-binaltorphimine (5 mg/kg i.p.) or the kappa-opioid receptor agonist U50,488H (1 mg/kg s.c.). Behavioural and endocrine experiments were performed in different groups of animals. Animals were exposed to the holeboard immediately followed by the plus-maze. Serum corticosterone levels were determined by radioimmunoassay. Nicotine induced an anxiogenic-like effect in the plus-maze and a significant decrease of holeboard activity. The anxiogenic-like effect in the plus-maze was not modified by any of the kappa-opioid receptor ligands. Nicotine also induced a significant increase in the corticosterone levels, and the kappa antagonist, which did not exert any effect per se, antagonised this effect. The kappa-agonist U50,488H induced a significant increase in corticosterone concentration when administered alone. We provide the first evidence for the involvement of the kappa-opioid receptor in the stimulatory effect of nicotine on adrenocortical activity.

  4. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  5. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT.

    Science.gov (United States)

    Zhan, Yifan; Gerondakis, Steve; Coghill, Elise; Bourges, Dorothee; Xu, Yuekang; Brady, Jamie L; Lew, Andrew M

    2008-10-15

    Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-kappaB pathway (RelA, cRel, and NF-kappaB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-kappaB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-kappaB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-kappaB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

  6. Kappa-opioid receptor-mediated effects of the plant-derived hallucinogen, salvinorin A, on inverted screen performance in the mouse.

    Science.gov (United States)

    Fantegrossi, William E; Kugle, Kelly M; Valdes, Leander J; Koreeda, Masato; Woods, James H

    2005-12-01

    Salvinorin A is a pharmacologically active diterpene that occurs naturally in the Mexican mint Ska Maria Pastora (Salvia divinorum) and represents the first naturally occurring kappa-opioid receptor agonist. The chemical structure of salvinorin A is novel among the opioids, and thus defines a new structural class of kappa-opioid-receptor selective drugs. Few studies have examined the effects of salvinorin A in vivo, and fewer still have attempted to assess the agonist actions of this compound at mu-opioid, delta-opioid, and kappa-opioid receptors using selective antagonists. In the mouse, salvinorin A disrupted climbing behavior on an inverted screen task, indicating a rapid, but short-lived induction of sedation/motor incoordination. Similar effects were observed with the mu-agonist remifentanil and the synthetic kappa-agonist U69,593. When behaviorally equivalent doses of all three opioids were challenged with antagonists at doses selective for mu-opioid, delta-opioid, or kappa-opioid receptors, results suggested that the motoric effects of remifentanil were mediated by mu-receptors, whereas those of salvinorin A and U69,593 were mediated via kappa-receptors. Despite similar potencies and degrees of effectiveness, salvinorin A and U69,593 differed with regard to their susceptibility to antagonism by the kappa-antagonist nor-binaltorphamine. This later finding, coupled with the novel chemical structure of the compound, is consistent with recent findings that the diterpene salvinorin A may bind to the kappa-receptor in a manner that is qualitatively different from that of more traditional kappa-agonists such as the benzeneacetamide U69,593. Such pharmacological differences among these kappa-opioids raise the possibility that the development of other diterpene-based opioids may yield important therapeutic compounds.

  7. Isolation and chemical modification of clerodane diterpenoids from Salvia species as potential agonists at the kappa-opioid receptor.

    Science.gov (United States)

    Li, Yiqiang; Husbands, Stephen M; Mahon, Mary F; Traynor, John R; Rowan, Michael G

    2007-07-01

    The clerodane diterpenoid salvinorin A (1), the main active component of the psychotropic herb Salvia divinorum, has been reported to be a potent agonist at the kappa-opioid receptor. Computer modeling suggested that splendidin (2) from S. splendens, as well as related compounds, might possess similar activities. In the present study, this hypothesis was tested by determination of the binding properties of a series of structural congeners, compounds 2-8, at the mu-, delta-, and kappa-opioid receptors. However, none of these compounds showed significant binding to any of the opioid-receptor subtypes, thus disproving the above hypothesis. The novel compounds 7 and 8 were obtained semi-synthetically by selective modification of salvifarin (5), isolated from Salvia farinacea, upon epoxide-ring opening with AcOH in the presence of indium(III) triflate. Also, the X-ray crystal structure of salvifaricin (6; Fig.), obtained from S. farinacea, was determined for the first time and used, in combination with in-depth NMR experiments, to elucidate the absolute configurations of the new products. Our experiments demonstrate that the relatively well-accessible diterpenoid 6 could be used as starting material for future studies into the structure-activity relationship at the kappa-opioid receptor.

  8. Antinociceptive profile of salvinorin A, a structurally unique kappa opioid receptor agonist.

    Science.gov (United States)

    McCurdy, Christopher R; Sufka, Kenneth J; Smith, Grant H; Warnick, Jason E; Nieto, Marcelo J

    2006-01-01

    Salvinorin A, is a structurally unique, non-nitrogenous, kappa opioid receptor (KOP) agonist. Given the role of KOPs in analgesic processes, we set out to determine whether salvinorin A has antinociceptive activity in thermal and chemo-nociceptive assays. The tail-flick assay was employed to investigate 1) salvinorin A's (0.5, 1.0, 2.0, and 4.0 mg/kg) dose-response and time-course (10, 20, and 30 min) effects in a thermal nociceptive assay, and 2) the ability for the KOP antagonist norBNI (10.0 mg/kg) to prevent salvinorin A antinociception. The hotplate assay was utilized as a second thermal nociceptive measure to test salvinorin A's dose-response effects. The acetic acid abdominal constriction assay was used to study salvinorin A's dose-response and time-course (over 30 min) effects in a chemo-nociceptive assay. Together, these studies revealed that salvinorin A produces a dose-dependent antinociception that peaked at 10 min post-injection but rapidly returned to baseline. Additionally, pretreatment with the KOP antagonist norbinaltorphimine (norBNI) reversed salvinorin A-induced antinociception. These findings demonstrate that salvinorin A produces a KOP mediated antinociceptive effect with a short duration of action.

  9. Receptor activator of nuclear factor kappa B (RANK as a determinant of peri-implantitis

    Directory of Open Access Journals (Sweden)

    Rakić Mia

    2013-01-01

    Full Text Available Background/Aim. Peri-implantitis presents inflammatory process that affects soft and hard supporting tissues of osseointegrated implant based on inflammatory osteoclastogenesis. The aim of this study was to investigate whether receptor activator of nuclear factor kappa B (RANK concentrations in peri-implant crevicular fluid could be associated with clinical parameters that reflect inflammatory nature of peri-implantitis. Methods. The study included 67 patients, 22 with diagnosed peri-implantitis, 22 persons with healthy peri-implant tissues and 23 patients with periodontitis. Clinical parameters from each patient were recorded and samples of peri-implant/gingival crevicular fluid were collected for the enzyme-linked immunosorbent assay (ELISA analysis. Results. RANK concentration was significantly increased in samples from the patients with periimplantitis when compared to healthy implants (p < 0.0001, where the average levels were 9 times higher. At the same time RANK concentration was significantly higher in periimplantitis than in periodontitis sites (p < 0.0001. In implant patients pocket depths and bleeding on probing values were positively associated with high RANK concentrations (p < 0.0001. Conclusion. These results revealed association of increased RANK concentration in samples of periimplant/ gingival crevicular fluid with peri-implant inflammation and suggests that RANK could be a pathologic determinant of peri-implantitis, thereby a potential parameter in assessment of peri-implant tissue inflammation and a potential target in designing treatment strategies.

  10. Inhibition of ErbB receptors, Hedgehog and NF-kappaB signaling by polyphenols in cancer.

    Science.gov (United States)

    Benvenuto, Monica; Fantini, Massimo; Masuelli, Laura; De Smaele, Enrico; Zazzeroni, Francesca; Tresoldi, Ilaria; Calabrese, Giorgio; Galvano, Fabio; Modesti, Andrea; Bei, Roberto

    2013-06-01

    Carcinogenesis is a multi-step process triggered by cumulative genetic alterations, which drive the progressive transformation of a normal cell into a cancer cell. Among the signal transduction pathways whose cross-talk plays an important role in neoplastic transformation are those mediated by ErbB receptors, NF-kappaB and the Hedgehog (HH)/glioma-associated oncogene (GLI) cascade. Polyphenols can be employed to inhibit the growth of cancer cells due to their ability to modulate the activity of multiple targets involved in carcinogenesis through simultaneous direct interaction or modulation of gene expression. This review will describe the cross-talk between ErbB receptors, NF-kappaB and the Hedgehog (HH)/glioma-associated oncogene (GLI) signaling pathways and the potential role of polyphenols in inhibiting this dialogue and the growth of cancer cells.

  11. Synthesis and characterization of a dual kappa-delta opioid receptor agonist analgesic blocking cocaine reward behavior.

    Science.gov (United States)

    Váradi, András; Marrone, Gina F; Eans, Shainnel O; Ganno, Michelle L; Subrath, Joan J; Le Rouzic, Valerie; Hunkele, Amanda; Pasternak, Gavril W; McLaughlin, Jay P; Majumdar, Susruta

    2015-11-18

    3-Iodobenzoyl naltrexamine (IBNtxA) is a potent analgesic belonging to the pharmacologically diverse 6β-amidoepoxymorphinan group of opioids. We present the synthesis and pharmacological evaluation of five analogs of IBNtxA. The scaffold of IBNtxA was modified by removing the 14-hydroxy group, incorporating a 7,8 double bond and various N-17 alkyl substituents. The structural modifications resulted in analogs with picomolar affinities for opioid receptors. The lead compound (MP1104) was found to exhibit approximately 15-fold greater antinociceptive potency (ED50 = 0.33 mg/kg) compared with morphine, mediated through the activation of kappa- and delta-opioid receptors. Despite its kappa agonism, this lead derivative did not cause place aversion or preference in mice in a place-conditioning assay, even at doses 3 times the analgesic ED50. However, pretreatment with the lead compound prevented the reward behavior associated with cocaine in a conditioned place preference assay. Together, these results suggest the promise of dual acting kappa- and delta-opioid receptor agonists as analgesics and treatments for cocaine addiction.

  12. Sex differences in kappa opioid receptor function and their potential impact on addiction

    Directory of Open Access Journals (Sweden)

    Elena eChartoff

    2015-12-01

    Full Text Available Behavioral, biological and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN, an endogenous ligand at kappa opioid receptors (KORs, is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain,mood and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN gene, genetic linkage with the melanocortin-1 receptor (MC1R, heterodimerization of KORs and mu opioid receptors (MORs, and gonadal hormones

  13. kappa+lambda+ dual receptor B cells are present in the human peripheral repertoire

    OpenAIRE

    1995-01-01

    It is a common notion that mature B lymphocytes express either kappa or lambda light (L) chains, although the mechanism that leads to such isotypic exclusion is still debated. We have investigated the extent of L chain isotypic exclusion in normal human peripheral blood B lymphocytes. By three-color staining with anti-CD19, anti-kappa, and anti-lambda antibodies we could estimate that 0.2-0.5% of peripheral blood B cells from healthy adults express both kappa and lambda on the cell surface. T...

  14. Kappa-opioid receptor antagonism improves recovery from myocardial stunning in chronically instrumented dogs.

    Science.gov (United States)

    Grosse Hartlage, Maike A; Theisen, Marc M; Monteiro de Oliveira, Nelson P; Van Aken, Hugo; Fobker, Manfred; Weber, Thomas P

    2006-10-01

    We tested the hypothesis that the selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI) improves recovery from myocardial stunning. Ten dogs were chronically instrumented for measurement of heart rate, left atrial, aortic and left ventricular pressure (LVP), and the maximum rate of LVP increase (LV dP/dt(max)) and decrease (LV dP/dt(max)), coronary blood flow velocity and myocardial wall-thickening fraction. Regional myocardial blood flow was determined with fluorescent microspheres. Catecholamine plasma levels were measured by high-performance liquid chromatography, and beta-endorphin and dynorphin plasma levels by radioimmunoassay. An occluder around the left anterior descending artery (LAD) allowed induction of a reversible LAD-ischemia. Animals underwent two experiments in a randomized crossover fashion on separate days: (a) 10 min LAD-occlusion (control experiment), (b) second ischemic episode 24 h after nor-BNI (2.5 mg/kg IV) (intervention). Dogs receiving nor-BNI showed an increase in wall-thickening fraction, LV dP/dt(max) and LV dP/dt(min) before ischemia and during the whole reperfusion (P < 0.05 versus control experiment). After nor-BNI pretreatment, dynorphin levels increased after induction of ischemia to a peak level of 15.1 +/- 3.6 pg/mL (P < 0.05 versus control experiment). The increase in plasma beta-endorphin during ischemia and early reperfusion was attenuated after nor-BNI. Compared with the control experiment, nor-BNI left global hemodynamics, regional myocardial blood flow, and catecholamine levels unchanged. In conclusion, nor-BNI improves recovery from myocardial stunning after regional myocardial ischemia in chronically instrumented dogs.

  15. Functional Selectivity of Kappa Opioid Receptor Agonists in Peripheral Sensory Neurons

    Science.gov (United States)

    Jamshidi, Raehannah J.; Jacobs, Blaine A.; Sullivan, Laura C.; Chavera, Teresa A.; Saylor, Rachel M.; Prisinzano, Thomas E.; Clarke, William P.

    2015-01-01

    Activation of kappa opioid receptors (KORs) expressed by peripheral sensory neurons that respond to noxious stimuli (nociceptors) can reduce neurotransmission of pain stimuli from the periphery to the central nervous system. We have previously shown that the antinociception dose-response curve for peripherally restricted doses of the KOR agonist (–)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50488) has an inverted U shape. Here, we found that the downward phase of the U50488 dose-response curve was blocked by an inhibitor of extracellular signal-regulated kinase (ERK) activation U0126. Local administration of the selective KOR agonist salvinorin A (Sal-A), also resulted in an inverted U-shaped curve; however, the downward phase was insensitive to U0126. By contrast, inhibition of c-Jun N-terminal kinase (JNK) partially blocked the downward phase of the dose-response curve to Sal-A, suggesting a role for JNK. In cultures of peripheral sensory neurons, U50488 and Sal-A inhibited adenylyl cyclase activity with similar efficacies; however, their ability to activate ERK and JNK differed. Whereas U50488 activated ERK but not JNK, Sal-A activated JNK but not ERK. Moreover, although both U50488 and Sal-A produced homologous desensitization, desensitization to U50488 was blocked by inhibition of ERK activation, whereas desensitization to Sal-A was blocked by inhibition of JNK. Substitution of an ethoxymethyl ether for the C2 position acetyl group of Sal-A reduced stimulation of JNK, prevented desensitization by ethoxymethyl ether for the C2 position acetyl group of Sal-A, and resulted in a monotonic antinociception dose-response curve. Collectively, these data demonstrate the functional selectivity of KOR ligands for signaling in peripheral sensory neurons, which results in differential effects on behavioral responses in vivo. PMID:26297384

  16. Caspase-8 regulation by direct interaction with TRAF6 in T cell receptor-induced NF-kappaB activation.

    Science.gov (United States)

    Bidère, Nicolas; Snow, Andrew L; Sakai, Keiko; Zheng, Lixin; Lenardo, Michael J

    2006-08-22

    Triggering of lymphocyte antigen receptors is the critical first step in the adaptive immune response against pathogens. T cell receptor (TCR) ligation assembles a large membrane signalosome, culminating in NF-kappaB activation [1,2]. Recently, caspase-8 was found to play a surprisingly prominent role in lymphocyte activation in addition to its well-known role in apoptosis [3]. Caspase-8 is activated after TCR stimulation and nucleates a complex with B cell lymphoma 10 (BCL10), paracaspase MALT1, and the inhibitors of kappaB kinase (IKK) complex [4]. We now report that the ubiquitin ligase TRAF6 binds to active caspase-8 upon TCR stimulation and facilitates its movement into lipid rafts. We identified in silico two putative TRAF6 binding motifs in the caspase-8 sequence and found that mutation of critical residues within these sites abolished TRAF6 binding and diminished TCR-induced NF-kappaB activation. Moreover, RNAi-mediated silencing of TRAF6 abrogated caspase-8 recruitment to the lipid rafts. Protein kinase Ctheta (PKCtheta), CARMA1, and BCL10 are also required for TCR-induced caspase-8 relocation, but only PKCtheta and BCL10 control caspase-8 activation. Our results suggest that PKCtheta independently controls CARMA1 phosphorylation and BCL10-dependent caspase-8 activation and unveil an essential role for TRAF6 as a critical adaptor linking these two convergent signaling events.

  17. Aberrant activation of the androgen receptor by NF-kappaB2/p52 in prostate cancer cells.

    Science.gov (United States)

    Nadiminty, Nagalakshmi; Lou, Wei; Sun, Meng; Chen, Jun; Yue, Jiao; Kung, Hsing-Jien; Evans, Christopher P; Zhou, Qinghua; Gao, Allen C

    2010-04-15

    Prostate cancer initiation and progression are uniquely dependent on the androgen receptor (AR). Even when the cancer progresses to a castration-resistant stage, AR signaling remains active via a variety of mechanisms. In the present study, we showed that NF-kappaB/p52 can activate the AR, resulting in increased transactivation of AR-responsive genes, such as PSA and NKX3.1, in a ligand-independent manner. NF-kappaB2/p52 enhances nuclear translocation and activation of AR by interacting with its NH(2)-terminal domain and enhances the recruitment of coactivators such as p300 to the promoters of AR-dependent genes. These results were confirmed in three different prostate cancer cell lines: LAPC-4 (wild-type AR), LNCaP (mutant AR), and C4-2 (castration resistant). Transfection of p52 into LAPC-4 and LNCaP cells (which express low levels of p52) showed increased activation of the endogenous AR. Downregulation of endogenous p52 in C4-2 cells resulted in abrogation of AR constitutive activation. Comparison of the relative effects of p52 and p65 (RelA) showed that p52, but not p65, could activate the AR. Collectively, these findings, together with previous reports that the levels of NF-kappaB2/p52 are elevated in prostate cancer cells and that active NF-kappaB2/p52 promotes prostate cancer cell growth in vitro and in vivo, suggest that NF-kappaB2/p52 may play a critical role in the progression of castration-resistant prostate cancer.

  18. New neoclerodane diterpenoids isolated from the leaves of Salvia divinorum and their binding affinities for human kappa opioid receptors.

    Science.gov (United States)

    Lee, David Y W; Ma, Zhongze; Liu-Chen, Lee-Yuan; Wang, Yulin; Chen, Yong; Carlezon, William A; Cohen, Bruce

    2005-10-01

    Bioactivity-guided fractionation of the leaves of Salvia divinorum has resulted in the isolation of three new neoclerodane diterpenoids: divinatorin D (1), divinatorin E (2), and salvinorin G (3), together with 10 known terpenoids, divinatorin C (4), hardwickiic acid (5), salvinorin-A (6), -B (7), -C (8), -D (9), -E (10), and -F (11), presqualene alcohol (12), and (E)-phytol (13). The structures of these three new compounds were characterized by spectroscopic methods. All these compounds were evaluated for their binding affinities to the human kappa opioid receptors. In comparison with divinatorin D (1), divinatorin E (2), and salvinorin G (3), salvinorin A (6) is still the most potent kappa agonist.

  19. Spironolactone induces apoptosis and inhibits NF-kappaB independent of the mineralocorticoid receptor

    DEFF Research Database (Denmark)

    Sønder, Søren Ulrik Salling; Woetmann, Anders; Odum, Niels

    2006-01-01

    10 and 1000 muM, respectively, significantly increased both apoptosis and cell death. Production of inflammatory cytokines was significantly reduced by 3 to 30 muM SPIR and by 300 to 1000 muM SFZ. We also found that 0.4 muM SPIR and 300 muM SFZ significantly reduced the activity of NF-kappa......B, a transcription factor involved in both apoptosis and immunoinflammation. ALDO, the MR antagonist, eplerenone, and the SPIR metabolite, 7alpha-thiomethyl-spironolactone, slightly reduced NF-kappaB activity, but they did not interfere with SPIR's effect, showing that MR binding is not involved in SPIR......-induced suppression of NF-kappaB activity. Finally, phosphorylation of IkappaBalpha was also significantly reduced by SPIR. These results provide new insight into the apoptotic and anti-inflammatory effects of SPIR....

  20. Kappa-opioid receptor signaling in the striatum as a potential modulator of dopamine transmission in cocaine dependence

    Directory of Open Access Journals (Sweden)

    Pierre eTrifilieff

    2013-06-01

    Full Text Available Cocaine addiction is accompanied by a decrease in striatal dopamine signaling, measured as a decrease in dopamine D2 receptor binding as well as blunted dopamine release in the striatum. These alterations in dopamine transmission have clinical relevance, and have been shown to correlate with cocaine-seeking behavior and response to treatment for cocaine dependence. However, the mechanisms contributing to the hypodopaminergic state in cocaine addiction remain unknown. Here we review the Positron Emission Tomography (PET imaging studies showing alterations in D2 receptor binding potential and dopamine transmission in cocaine abusers and their significance in cocaine-seeking behavior. Based on animal and human studies, we propose that the kappa receptor/dynorphin system, because of its impact on dopamine transmission and upregulation following cocaine exposure, could contribute to the hypodopaminergic state reported in cocaine addiction, and could thus be a relevant target for treatment development.

  1. Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

    Institute of Scientific and Technical Information of China (English)

    JIE CAO; PING ZHAO; X1AO HUI MIAO; LAN JUAN ZHAO; LI JUN XUE; ZHONG TIAN QI

    2003-01-01

    The human CD81(hCD81),the most recently proposed receptor of hepatitis C virus(HCV),can especifically bind to HCV envelope glycoprotein 2(E2).In this study,hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library(PVIII9aaCys).Eighteen of the 75clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay(ELISA)and competitive inhibition test.Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA.Sequence comparison of the motif showed no amino acid homology with the native HCV E2.The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells,and induce HCV E2 specific immune response in vivo.These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules.Our findings cast new light on developing HCV receptor antagonists.

  2. Expression of {mu}, {kappa}, and {delta} opioid receptor messenger RNA in the human CNS: a {sup 33}P in situ hybridization study

    Energy Technology Data Exchange (ETDEWEB)

    Peckys, D.; Landwehrmeyer, G.B. [Department of Neurology, Albert-Ludwigs-University Freiburg, Neurozentrum, Breisacherstrasse 64, D-79106 Freiburg (Germany)

    1999-02-01

    The existence of at least three opioid receptor types, referred to as {mu}, {kappa}, and {delta}, is well established. Complementary DNAs corresponding to the pharmacologically defined {mu}, {kappa}, and {delta} opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using {sup 33}P-labelled RNA probes. In the present study we report the regional and cellular expression of {mu}, {kappa}, and {delta} opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the {mu} and {kappa} opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the {delta} opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of {mu}, {kappa}, and {delta} opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between {mu} opioid receptor receptor binding and {mu} opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous

  3. Salvinorin A, an active component of the hallucinogenic sage salvia divinorum is a highly efficacious kappa-opioid receptor agonist: structural and functional considerations.

    Science.gov (United States)

    Chavkin, Charles; Sud, Sumit; Jin, Wenzhen; Stewart, Jeremy; Zjawiony, Jordan K; Siebert, Daniel J; Toth, Beth Ann; Hufeisen, Sandra J; Roth, Bryan L

    2004-03-01

    The diterpene salvinorin A from Salvia divinorum has recently been reported to be a high-affinity and selective kappa-opioid receptor agonist (Roth et al., 2002). Salvinorin A and selected derivatives were found to be potent and efficacious agonists in several measures of agonist activity using cloned human kappa-opioid receptors expressed in human embryonic kidney-293 cells. Thus, salvinorin A, salvinorinyl-2-propionate, and salvinorinyl-2-heptanoate were found to be either full (salvinorin A) or partial (2-propionate, 2-heptanoate) agonists for inhibition of forskolin-stimulated cAMP production. Additional studies of agonist potency and efficacy of salvinorin A, performed by cotransfecting either the chimeric G proteins Gaq-i5 or the universal G protein Ga16 and quantification of agonist-evoked intracellular calcium mobilization, affirmed that salvinorin A was a potent and effective kappa-opioid agonist. Results from structure-function studies suggested that the nature of the substituent at the 2-position of salvinorin A was critical for kappa-opioid receptor binding and activation. Because issues of receptor reserve complicate estimates of agonist efficacy and potency, we also examined the agonist actions of salvinorin A by measuring potassium conductance through G protein-gated K(+) channels coexpressed in Xenopus oocytes, a system in which receptor reserve is minimal. Salvinorin A was found to be a full agonist, being significantly more efficacious than (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50488) or (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593) (two standard kappa-opioid agonists) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for kappa-opioid receptors). Salvinorin A thus represents the first known naturally occurring non-nitrogenous full agonist at kappa-opioid receptors.

  4. PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation.

    Science.gov (United States)

    Tanaka, Hiroshi; Hoshikawa, Yutaka; Oh-hara, Tomoko; Koike, Sumie; Naito, Mikihiko; Noda, Tetsuo; Arai, Hiroyuki; Tsuruo, Takashi; Fujita, Naoya

    2009-04-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-alpha-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of kappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB), leading to induction of several NF-kappaB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-alpha, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-alpha-mediated NF-kappaB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKbeta, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-kappaB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments

  5. The isolation of novel phage display-derived human recombinant antibodies against CCR5, the major co-receptor of HIV.

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

    2013-08-01

    Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.

  6. Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

    Science.gov (United States)

    2011-09-28

    including the possible use of bacteriophages [5]. There are more and more publications on successful bacteriophage treatment of various experimental and...Discov Today 14: 536–540. 7. Deresinski S (2009) Bacteriophage therapy: exploiting smaller fleas . Clin Infect Dis 48: 1096–1101. 8. Kutter E, De Vos D...PCR. PLoS ONE 5(6): e11337. 28. Smith HW, Huggins MB (1983) Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves

  7. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    Science.gov (United States)

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  8. Immediate and Persistent Effects of Salvinorin A on the Kappa Opioid Receptor in Rodents, Monitored In Vivo with PET.

    Science.gov (United States)

    Placzek, Michael S; Van de Bittner, Genevieve C; Wey, Hsiao-Ying; Lukas, Scott E; Hooker, Jacob M

    2015-12-01

    Monitoring changes in opioid receptor binding with positron emission tomography (PET) could lead to a better understanding of tolerance and addiction because altered opioid receptor dynamics following agonist exposure has been linked to tolerance mechanisms. We have studied changes in kappa opioid receptor (KOR) binding availability in vivo with PET following kappa opioid agonist administration. Male Sprague-Dawley rats (n=31) were anesthetized and treated with the (KOR) agonist salvinorin A (0.01-1.8 mg/kg, i.v.) before administration of the KOR selective radiotracer [(11)C]GR103545. When salvinorin A was administered 1 min prior to injection of the radiotracer, [(11)C]GR103545 binding potential (BPND) was decreased in a dose-dependent manner, indicating receptor binding competition. In addition, the unique pharmacokinetics of salvinorin A (half-life ~8 min in non-human primates) allowed us to study the residual impact on KOR after the drug had eliminated from the brain. Salvinorin A was administered up to 5 h prior to [(11)C]GR103545, and the changes in BPND were compared with baseline, 2.5 h, 1 h, and 1 min pretreatment times. At lower doses (0.18 mg/kg and 0.32 mg/kg) we observed no prolonged effect on KOR binding but at 0.60 mg/kg salvinorin A induced a sustained decrease in KOR binding (BPND decreased by 40-49%) which persisted up to 2.5 h post administration, long after salvinorin A had been eliminated from the brain. These data point towards an agonist-induced adaptive response by KOR, the dynamics of which have not been previously studied in vivo with PET.

  9. The Differential Effects of a Selective Kappa-Opioid Receptor Agonist, U50488, in Guinea Pig Heart Tissues

    Directory of Open Access Journals (Sweden)

    Chi-Feng Hung

    2015-01-01

    Full Text Available The differential effects of a selective kappa- (κ- opioid receptor agonist, U50488, were elucidated by monitoring the contraction of isolated guinea pig atrial and ventricular muscles. In electrically driven left atria, U50488 in nanomolar concentration range decreased the contractile force. Norbinaltorphimine (norBNI, a selective κ-receptor antagonist, and pertussis toxin (PTX abolished the negative inotropic effect of U50488. In contrast, the inhibitory effect was not affected by the pretreatment of atropine or propranolol. Even though U50488 exerted a negative inotropic effect in the left atrium, it did not affect the contractile force of the right atrium and ventricles paced at 2 Hz. Similarly, the beating rate of the spontaneously beating right atrium was also unaffected by U50488. These results indicate that the activation of κ-opioid receptors can only produce negative inotropic effect in left atria via activation of PTX-sensitive G protein in guinea pigs. The absence of negative inotropic effects in right atria and ventricles suggests that there may be a greater distribution of functional κ-opioid receptors in guinea pig left atria than in right atria and ventricles, and the distribution of the receptors may be species-specific.

  10. Synthesis and pharmacological evaluation of [(3)H]HS665, a novel, highly selective radioligand for the kappa opioid receptor.

    Science.gov (United States)

    Guerrieri, Elena; Mallareddy, Jayapal Reddy; Tóth, Géza; Schmidhammer, Helmut; Spetea, Mariana

    2015-03-18

    Herein we report the radiolabeling and pharmacological investigation of a novel radioligand, the N-cyclobutylmethyl substituted diphenethylamine [(3)H]HS665, designed to bind selectively to the kappa opioid peptide (KOP) receptor, a target of therapeutic interest for the treatment of a variety of human disorders (i.e., pain, affective disorders, drug addiction, and psychotic disorders). HS665 was prepared in tritium-labeled form by a dehalotritiated method resulting in a specific activity of 30.65 Ci/mmol. Radioligand binding studies were performed to establish binding properties of [(3)H]HS665 to the recombinant human KOP receptor in membranes from Chinese hamster ovary cells stably expressing human KOP receptors (CHOhKOP) and to the native neuronal KOP receptor in guinea pig brain membranes. Binding of [(3)H]HS665 was specific and saturable in both tissue preparations. A single population of high affinity binding sites was labeled by [(3)H]HS665 in membranes from CHOhKOP cells and guinea pig brain with similar equilibrium dissociation constants, Kd, 0.45 and 0.64 nM, respectively. Average receptor density of [(3)H]HS665 recognition sites were 5564 and 154 fmol/mg protein in CHOhKOP cells and guinea pig brain, respectively. This study shows that the new radioligand distinguishes and labels KOP receptors specifically in neuronal and cellular systems expressing KOP receptors, making this molecule a valuable tool in probing structural and functional mechanisms governing ligand-KOP receptor interactions in both a recombinant and native in vitro setting.

  11. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4.

    Science.gov (United States)

    Newton, Kim; Sun, Xiaoqing; Dixit, Vishva M

    2004-02-01

    RIP3 is a member of the RIP kinase family. It is expressed in the embryo and in multiple adult tissues, including most hemopoietic cell lineages. Several studies have implicated RIP3 in the regulation of apoptosis and NF-kappa B signaling, but whether RIP3 promotes or attenuates activation of the NF-kappa B family of transcription factors has been controversial. We have generated RIP3-deficient mice by gene targeting and find RIP3 to be dispensable for normal mouse development. RIP3-deficient cells showed normal sensitivity to a variety of apoptotic stimuli and were indistinguishable from wild-type cells in their ability to activate NF-kappa B signaling in response to the following: human tumor necrosis factor (TNF), which selectively engages mouse TNF receptor 1; cross-linking of the B- or T-cell antigen receptors; peptidoglycan, which activates Toll-like receptor 2; and lipopolysaccharide (LPS), which stimulates Toll-like receptor 4. Consistent with these observations, RIP3-deficient mice exhibited normal antibody production after immunization with a T-dependent antigen and normal interleukin-1 beta (IL-1 beta), IL-6, and TNF production after LPS treatment. Thus, we can exclude RIP3 as an essential modulator of NF-kappa B signaling downstream of several receptor systems.

  12. Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin A ligands.

    Science.gov (United States)

    Polepally, Prabhakar R; White, Kate; Vardy, Eyal; Roth, Bryan L; Ferreira, Daneel; Zjawiony, Jordan K

    2013-05-15

    Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand-receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ-, δ- and μ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (Ki=2nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (Ki=21, 36 and 39nM).

  13. Inhibition of trigemino-hypoglossal reflex in rats by oxytocin is mediated by mu and kappa opioid receptors.

    Science.gov (United States)

    Zubrzycka, Maria; Fichna, Jakub; Janecka, Anna

    2005-02-21

    Recent studies showed that oxytocin plays an important role in the modulation of pain at different levels of the central nervous system. The present study was undertaken to investigate the effect of oxytocin on trigemino-hypoglossal reflex in rats. With the experimental settings used in this study, we have demonstrated that oxytocin showed significant analgesic effect after intracerebroventricular administration in rats, as assayed by the amplitude of the retractory movements of the tongue after tooth pulp stimulation. Antinociceptive effect of oxytocin was inhibited by subsequent perfusion of cerebral ventricles with oxytocin antagonist, [deamino-Cys1-D-Tyr(OEt)2-Thr4-Orn8]-oxytocin, atosiban. An involvement of opioid system in the oxytocin-induced analgesia was studied after intracerebroventricular administration of different opioid antagonists: non-selective naloxone, mu-selective beta-funaltrexamine, delta-selective naltrindole, and kappa-selective nor-binaltorphimine. It was shown that inhibition of antinociceptive effects was mediated through mu and kappa opioid receptors, indicating that there is a synergy between oxytocin and opioid systems in transmitting and modulating pain stimuli. Co-administration of oxytocin and a mu-selective endogenous opioid ligand endomorphin-2 did not significantly increase the antinociceptive activity of endomorphin-2.

  14. Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha.

    Science.gov (United States)

    Kim, Hyon Jong; Chang, Eun-Ju; Kim, Hyun-Man; Lee, Seung Bok; Kim, Hyun-Duck; Su Kim, Ghi; Kim, Hong-Hee

    2006-05-01

    The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.

  15. Toll-like receptor 4/nuclear factor-kappa B signaling detected in brain after early subarachnoid hemorrhage

    Institute of Scientific and Technical Information of China (English)

    MA Chun-xiao; YIN Wei-ning; CAI Bo-wen; WU Jian; WANG Jun-yi; HE Min; SUN Hong; DING Jun-li; YOU Chao

    2009-01-01

    Background Inflammation and immunity play a vital role in the pathogenesis of early brain injury after subarachnoid hemorrhage (SAH). Nuclear factor-kappa B (NF-KB) regulates many genes essential for inflammation and immunity and is activated by toll-like receptor (TLR). This study aimed to detect the expression of the toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-KB) signaling in the rat brain after early SAH. Methods The rats were decapitated and their brains were removed at 0, 2, 4, 6, 12, 24 and 48 hours after a single injection of blood into the prechiasmatic cistern, mRNA expression of TLR4 was measured by Taqman real-time RT-PCR, and protein expression by immunohistochemistry and Western blotting. NF-KB activity and concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-lbeta (IL-β) and intedeukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA).Results TaqMan real-time RT-PCR and Western blotting identified a biphasic change in TLR4 expression in both mRNA and protein: an initial peak (2-6 hours) and a sustained elevation (12-48 hours). Immunohistochemical staining showed the inducible expression of TLR4-1ike immunoreactions predominantly in glial cells and vascular endothelium. A similar biphasic change in the activation of NF-KB subunit p65 as well as the production of NF-KB-regulated proinflammatory cytokines (TNF-α, IL-1β and IL-6) were detected by ELISA. Conclusions These data suggest that experimental SAH induces significant up-regulation of TLR4 expression and the NF-KB signaling in early brain injury. Activation of the TLR4/NF-KB signaling may regulate the inflammatory responses after SAH.

  16. Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

    Science.gov (United States)

    Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

    2016-01-01

    Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

  17. Inhibition of receptor activator of NF-kappaB ligand by denosumab attenuates vascular calcium deposition in mice.

    Science.gov (United States)

    Helas, Susann; Goettsch, Claudia; Schoppet, Michael; Zeitz, Ute; Hempel, Ute; Morawietz, Henning; Kostenuik, Paul J; Erben, Reinhold G; Hofbauer, Lorenz C

    2009-08-01

    Osteoporosis and vascular calcification frequently coincide. A potential mediator of bone metabolism and vascular homeostasis is the triad cytokine system, which consists of receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL), its receptor RANK, and the decoy receptor osteoprotegerin. Unopposed RANKL activity in osteoprotegerin-deficient mice resulted in osteoporosis and vascular calcification. We therefore analyzed the effects of RANKL inhibition by denosumab, a human monoclonal antibody against RANKL, on vascular calcium deposition following glucocorticoid exposure. Prednisolone pellets were implanted into human RANKL knock-in (huRANKL-KI) mice, which unlike wild-type mice are responsive to denosumab. No histomorphological abnormalities or differences in aortic wall thickness were detected between wild-type and huRANKL-KI mice, regardless of treatment with prednisolone, denosumab, or both. However, concurrent treatment with denosumab reduced aortic calcium deposition of prednisolone-treated huRANKL-KI mice by up to 50%, based on calcium measurement. Of note, aortic calcium deposition in huRANKL-KI mice was correlated negatively with bone mineral density at the lumbar spine (P = 0.04) and positively with urinary excretion of deoxypyridinoline, a marker of bone resorption (P = 0.01). In summary, RANKL inhibition by denosumab reduced vascular calcium deposition in glucocorticoid-induced osteoporosis in mice, which is further evidence for the link between the bone and vascular systems. Therefore, the prevention of bone loss by denosumab might also be associated with reduced vascular calcification in certain conditions.

  18. Phage therapy pharmacology phage cocktails.

    Science.gov (United States)

    Chan, Benjamin K; Abedon, Stephen T

    2012-01-01

    Phage therapy is the clinical or veterinary application of bacterial viruses (bacteriophages) as antibacterial "drugs." More generally, phages can be used as biocontrol agents against plant as well as foodborne pathogens. In this chapter, we consider the therapeutic use of phage cocktails, which is the combining of two or more phage types to produce more pharmacologically diverse formulations. The primary motivation for the use of cocktails is their broader spectra of activity in comparison to individual phage isolates: they can impact either more bacterial types or achieve effectiveness under a greater diversity of conditions. The combining of phages can also facilitate better targeting of multiple strains making up individual bacterial species or covering multiple species that might be responsible for similar disease states, in general providing, relative to individual phage isolates, a greater potential for presumptive or empirical treatment. Contrasting the use of phage banks, or even phage isolation against specific etiologies that have been obtained directly from patients under treatment, here we consider the utility as well as potential shortcomings associated with the use of phage cocktails as therapeutic antibacterial agents.

  19. Biological variation and reference intervals for circulating osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor kappa B ligand and high-sensitivity C-reactive protein

    DEFF Research Database (Denmark)

    Sennels, H P; Jacobsen, Søren; Jensen, T

    2007-01-01

    Objective. Monitoring inflammatory diseases and osteoclastogenesis with osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor kappa B ligand (total sRANKL) and high-sensitivity C-reactive protein (hsCRP) has recently attracted increased interest. The purpose...

  20. Receptor-selective changes in mu-, delta- and kappa-opioid receptors after chronic naltrexone treatment in mice

    NARCIS (Netherlands)

    Lesscher, HMB; Bailey, Alexis; Burbach, JPH; van Ree, JM; Kitchen, [No Value; Gerrits, MAFM

    2003-01-01

    Chronic treatment with the opioid antagonist naltrexone induces functional supersensitivity to opioid agonists, which may be explained by receptor up-regulation induced by opioid receptor blockade. In the present study, the levels of opioid receptor subtypes through the brain of mice were determined

  1. Receptor-selective changes in mu-, delta- and kappa-opioid receptors after chronic naltrexone treatment in mice

    NARCIS (Netherlands)

    Lesscher, HMB; Bailey, Alexis; Burbach, JPH; van Ree, JM; Kitchen, [No Value; Gerrits, MAFM

    Chronic treatment with the opioid antagonist naltrexone induces functional supersensitivity to opioid agonists, which may be explained by receptor up-regulation induced by opioid receptor blockade. In the present study, the levels of opioid receptor subtypes through the brain of mice were determined

  2. Indoxyl Sulfate Downregulates Mas Receptor via Aryl Hydrocarbon Receptor/Nuclear Factor-kappa B, and Induces Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Ng, Hwee-Yeong; Bolati, Wulaer; Lee, Chien-Te; Chien, Yu-Shu; Yisireyili, Maimaiti; Saito, Shinichi; Pei, Sung-Nan; Nishijima, Fuyuhiko; Niwa, Toshimitsu

    2016-01-01

    Angiotensin converting enzyme-related carboxypeptidase 2/angiotensin (Ang)-(1-7)/Mas receptor axis is protective in the development of chronic kidney disease and cardiovascular disease. This study is aimed at investigating whether indoxyl sulfate (IS) affects Mas receptor expression, cell proliferation and tissue factor expression in vascular smooth muscle cells, and if Ang-(1-7), an activator of Mas receptor, counteracts the IS-induced effects. IS was administered to normotensive and hypertensive rats. Human aortic smooth muscle cells (HASMCs) were cultured with IS. IS reduced the expression of Mas receptor in the aorta of normotensive and hypertensive rats. IS downregulated the Mas receptor expression in a time- and dose-dependent manner in HASMCs. Knockdown of aryl hydrocarbon receptor (AhR) and nuclear factor-kappa B (NF-x03BA;B) inhibited IS-induced downregulation of Mas receptor. Further, IS stimulated cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) attenuated IS-induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) suppressed phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-x03BA;B in HASMCs. IS downregulated the expression of Mas receptor via AhR/NF-x03BA;B, and induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) inhibited IS-induced cell proliferation and tissue factor expression by suppressing the phosphorylation of ERK1/2 and NF-x03BA;B p65. © 2016 S. Karger AG, Basel.

  3. Salvinorin A inhibits colonic transit and neurogenic ion transport in mice by activating kappa-opioid and cannabinoid receptors.

    Science.gov (United States)

    Fichna, J; Schicho, R; Andrews, C N; Bashashati, M; Klompus, M; McKay, D M; Sharkey, K A; Zjawiony, J K; Janecka, A; Storr, M A

    2009-12-01

    The major active ingredient of the plant Salvia divinorum, salvinorin A (SA) has been used to treat gastrointestinal (GI) symptoms. As the action of SA on the regulation of colonic function is unknown, our aim was to examine the effects of SA on mouse colonic motility and secretion in vitro and in vivo. The effects of SA on GI motility were studied using isolated preparations of colon, which were compared with preparations from stomach and ileum. Colonic epithelial ion transport was evaluated using Ussing chambers. Additionally, we studied GI motility in vivo by measuring colonic propulsion, gastric emptying, and upper GI transit. Salvinorin A inhibited contractions of the mouse colon, stomach, and ileum in vitro, prolonged colonic propulsion and slowed upper GI transit in vivo. Salvinorin A had no effect on gastric emptying in vivo. Salvinorin A reduced veratridine-, but not forskolin-induced epithelial ion transport. The effects of SA on colonic motility in vitro were mediated by kappa-opioid receptors (KORs) and cannabinoid (CB) receptors, as they were inhibited by the antagonists nor-binaltorphimine (KOR), AM 251 (CB(1) receptor) and AM 630 (CB(2) receptor). However, in the colon in vivo, the effects were largely mediated by KORs. The effects of SA on veratridine-mediated epithelial ion transport were inhibited by nor-binaltorphimine and AM 630. Salvinorin A slows colonic motility in vitro and in vivo and influences neurogenic ion transport. Due to its specific regional action, SA or its derivatives may be useful drugs in the treatment of lower GI disorders associated with increased GI transit and diarrhoea.

  4. Oxidative stress causes renal dopamine D1 receptor dysfunction and hypertension via mechanisms that involve nuclear factor-kappaB and protein kinase C.

    Science.gov (United States)

    Banday, Anees Ahmad; Fazili, Fatima Rizwan; Lokhandwala, Mustafa F

    2007-05-01

    Renal dopamine, via activation of D1 receptors, plays a role in maintaining sodium homeostasis and BP. There exists a defect in renal D1 receptor function in hypertension, diabetes, and aging, conditions that are associated with oxidative stress. However, the exact underlying mechanism of the oxidative stress-mediated impaired D1 receptor signaling and hypertension is not known. The effect of oxidative stress on renal D1 receptor function was investigated in healthy animals. Male Sprague-Dawley rats received tap water (vehicle) and 30 mM L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mM tempol for 2 wk. Compared with vehicle, BSO treatment caused oxidative stress and increase in BP, which was accompanied by defective D1 receptor G-protein coupling and loss of natriuretic response to SKF38393. BSO treatment also increased NF-kappaB nuclear translocation, protein kinase C (PKC) activity and expression, G-protein-coupled receptor kinase-2 (GRK-2) membranous translocation, and D1 receptor serine phosphorylation. In BSO-treated rats' supplementation of tempol decreased oxidative stress, normalized BP, and restored D1 receptor G-protein coupling and natriuretic response to SKF38393. Tempol also normalized NF-kappaB translocation, PKC activity and expression, GRK-2 sequestration, and D1 receptor serine phosphorylation. In conclusion, these results show that oxidative stress activates NF-kappaB, causing an increase in PKC activity, which leads to GRK-2 translocation and subsequent D1 receptor hyper-serine phosphorylation and uncoupling. The functional consequence of this phenomenon was the inability of SKF38393 to inhibit Na/K-ATPase activity and promote sodium excretion, which may have contributed to increase in BP. Tempol reduced oxidative stress and thereby restored D1 receptor function and normalized BP.

  5. Pharmacological and genetic manipulation of kappa opioid receptors: effects on cocaine- and pentylenetetrazol-induced convulsions and seizure kindling.

    Science.gov (United States)

    Kaminski, Rafal M; Witkin, Jeffrey M; Shippenberg, Toni S

    2007-03-01

    The present study used pharmacological and gene ablation techniques to examine the involvement of kappa opioid receptors (KOPr) in modulating the convulsant effects of two mechanistically different drugs: cocaine and pentylenetetrazol (PTZ; GABA-A receptor antagonist) in mice. Systemic administration of the selective KOPr-1 agonist, U69593 (0.16-0.6mg/kg; s.c.), failed to modify cocaine-evoked convulsions or cocaine kindling. Similarly, no alteration in responsiveness to cocaine was observed in wild-type mice that received the selective KOPr-1 antagonist, nor-binaltorphimine (nor-BNI; 5mg/kg) or in mice lacking the gene encoding KOPr-1. In contrast to cocaine, U69593 attenuated the seizures induced by acute or repeated PTZ administration. Nor-BNI decreased the threshold for PTZ-evoked seizures and increased seizure incidence during the initial induction of kindling relative to controls. Decreased thresholds for PTZ-induced seizures were also observed in KOPr-1 knock out mice. Together, these data demonstrate an involvement of endogenous KOPr systems in modulating vulnerability to the convulsant effects of PTZ but not cocaine. Furthermore, they demonstrate that KOPr-1 activation protects against acute and kindled seizures induced by this convulsant. Finally, the results of our study suggest that KOPr-1 antagonists will not have therapeutic utility against cocaine-induced seizures, while they may prove beneficial in attenuating several actions of cocaine that have been linked to its abuse.

  6. Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity. Studies with salvinorin A and the kappa-opioid receptor.

    Science.gov (United States)

    Vortherms, Timothy A; Mosier, Philip D; Westkaemper, Richard B; Roth, Bryan L

    2007-02-02

    Salvinorin A, the active component of the hallucinogenic sage Salvia divinorum, is an apparently selective and highly potent kappa-opioid receptor (KOR) agonist. Salvinorin A is unique among ligands for peptidergic G protein-coupled receptors in being nonnitrogenous and lipid-like in character. To examine the molecular basis for the subtype-selective binding of salvinorin A, we utilized an integrated approach using chimeric opioid receptors, site-directed mutagenesis, the substituted cysteine accessibility method, and molecular modeling and dynamics studies. We discovered that helix 2 is required for salvinorin A binding to KOR and that two residues (Val-108(2.53) and Val-118(2.63)) confer subtype selectivity. Intriguingly, molecular modeling studies predicted that these loci exhibit an indirect effect on salvinorin A binding, presumably through rotation of helix 2. Significantly, and in agreement with our in silico predictions, substituted cysteine accessibility method analysis of helix 2 comparing KOR and the delta-opioid receptor, which has negligible affinity for salvinorin A, revealed that residues known to be important for salvinorin A binding exhibit a differential pattern of water accessibility. These findings imply that differences in the helical orientation of helix 2 are critical for the selectivity of salvinorin A binding to KOR and provide a structurally novel basis for ligand selectivity.

  7. Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

    Science.gov (United States)

    Aksoy, Ezra; Vanden Berghe, Wim; Detienne, Sophie; Amraoui, Zoulikha; Fitzgerald, Kathrine A; Haegeman, Guy; Goldman, Michel; Willems, Fabienne

    2005-07-01

    Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)-mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF)-dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappaB, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IkappaB kinase (IKK)-alpha/beta phosphorylation and IkappaB-alpha degradation with a concomitant increase in NF-kappaB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappaB-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappaB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappaB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-kappaB activity, and thereby increases IFN-beta synthesis elicited by TLR3 or TLR4 ligands.

  8. Identification of the molecular mechanisms by which the diterpenoid salvinorin A binds to kappa-opioid receptors.

    Science.gov (United States)

    Yan, Feng; Mosier, Philip D; Westkaemper, Richard B; Stewart, Jeremy; Zjawiony, Jordan K; Vortherms, Timothy A; Sheffler, Douglas J; Roth, Bryan L

    2005-06-21

    Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates kappa-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.

  9. Salvinorin A administration after global cerebral hypoxia/ischemia preserves cerebrovascular autoregulation via kappa opioid receptor in piglets.

    Directory of Open Access Journals (Sweden)

    Zhenhong Wang

    Full Text Available BACKGROUND: Cerebral hypoxia/ischemia (HI is not uncommon during the perinatal period. If occurring, it can result in severe neurologic disabilities that persist throughout life. Salvinorin A, a non-opioid Kappa opioid receptors (KOR selective agonist, has the potential to address this devastating situation. We have demonstrated that salvinorin A administration before HI, preserves pial artery autoregulative function through both the KOR and extracellular signal-regulated kinases (ERK pathways. In the present study, we tested the hypothesis that administration of salvinorin A after HI could preserve cerebral autoregulation via KOR and ERK pathway. METHODOLOGY/PRINCIPAL FINDINGS: The response of the pial artery to hypercapnia, hypotension and isoproterenol were monitored before and 1 hour after HI in piglets equipped with a cranial window. Four groups of drug administration were performed after HI. The control group had DMSO (1 µl/kg, i.v. administrated immediately after HI. Two salvinorin A treated groups had salvinorin A (10 µg/kg, i.v. administrated 0 and 30 min after HI, respectively. The 4(th group had salvinorin A and the KOR antagonist norbinaltorphimine (Nor-BIN, 1 µM topical co-administrated 0 min after HI (n = 5. The dilation responses of the pial artery to hypercapnia and hypotension were impaired after global HI and were preserved with salvinorin A administration immediately or 30 min after HI. The preservation of autoregulation was abolished when nor-BIN was administered. Levels of phosphor-ERK(pERK/ERK in the cerebrospinal fluid (CSF were measured before and 1 hour after HI. After HI, the pERK/ERK levels significantly increased in both DMSO control group and salvinorin A and nor-BIN co-administration group. The elevated levels of pERK/ERK were not observed with salvinorin A only groups. CONCLUSIONS: Salvinorin A administration 0 and 30 min after HI preserves autoregulation of pial artery to hypercapnia and hypotension via

  10. TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

    Science.gov (United States)

    Pype, S; Declercq, W; Ibrahimi, A; Michiels, C; Van Rietschoten, J G; Dewulf, N; de Boer, M; Vandenabeele, P; Huylebroeck, D; Remacle, J E

    2000-06-16

    CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

  11. Two flagellotropic phages and one pilus-specific phage active against Asticcacaulis biprosthecum.

    Science.gov (United States)

    Pate, J L; Petzold, S J; Umbreit, T H

    1979-04-15

    Three phages active against cells of Asticcacaulis biprosthecum attach to receptor sites located at the pole of the cell where pili, flagella, and holdfast are produced. Phage phiAcS2, a large phage with a prolate cylindrical head and flexible, noncontractile tail, attaches to flagella as well as to receptor sites at the pole of the cell. Attachment to flagella occurs at the region where head and tail of the phage are joined, leaving the distal end of the tail free for attachment to receptor sites at the cell surface. Phages phiAcM2 and phiAcM4, are identical in appearance to each other, possessing prolate cylindrical heads and flexible, noncontractile tails, and are smaller than phage phiAcS2. Phage phiAcM4, exhibits the same flagellotropic characteristic as described for phage phiAcS2, including the manner of attachment to flagella. Phage phiAcM2 has no affinity for flagella, but attaches by the distal end of the tail to pili and to receptor sites at the pole of the cell. Mechanical removal of flagella and pili protects against infection by all three phages. Studies with phage-resistant mutants and with KCN-treated cells suggest that pili are required for infection by both flagellotropic and pilus-specific phages.

  12. A novel, potent, oral active and safe antinociceptive pyrazole targeting kappa opioid receptors.

    Science.gov (United States)

    Trevisan, Gabriela; Rossato, Mateus F; Walker, Cristiani I B; Oliveira, Sara M; Rosa, Fernanda; Tonello, Raquel; Silva, Cássia R; Machado, Pablo; Boligon, Aline A; Martins, Marcos A P; Zanatta, Nilo; Bonacorso, Hélio G; Athayde, Margareth L; Rubin, Maribel A; Calixto, João B; Ferreira, Juliano

    2013-10-01

    Pyrazole compounds are an intriguing class of compounds with potential analgesic activity; however, their mechanism of action remains unknown. Thus, the goal of this study was to explore the antinociceptive potential, safety and mechanism of action of novel 1-pyrazole methyl ester derivatives, which were designed by molecular simplification, using in vivo and in vitro methods in mice. First, tree 1-pyrazole methyl ester derivatives (DMPE, MPFE, and MPCIE) were tested in the capsaicin test and all presented antinociceptive effect; however the MPClE (methyl 5-trichloromethyl-3-methyl-1H-pyrazole-1-carboxylate) was the most effective. Thus, we selected this compound to assess the effects and mechanisms in subsequent pain models. MPCIE produced antinociception when administered by oral, intraperitoneal, intrathecal and intraplantar routes and was effective in the capsaicin and the acetic acid-induced nociception tests. Moreover, this compound reduced the hyperalgesia in diverse clinically-relevant pain models, including postoperative, inflammatory, and neuropathic nociception in mice. The antinociception produced by orally administered MPClE was mediated by κ-opioid receptors, since these effects were prevented by systemically pre-treatment with naloxone and the κ-opioid receptor antagonist nor-binaltorphimine. Moreover, MPCIE prevented binding of the κ-opioid ligand [(3)H]-CI-977 in vitro (IC₅₀ of 0.68 (0.32-1.4) μM), but not the TRPV1 ([(3)H]-resiniferatoxin) or the α₂-adrenoreceptor ([(3)H]-idazoxan) binding. Regarding the drug-induced side effects, oral administration of MPClE did not produce sedation, constipation or motor impairment at its active dose. In addition, MPCIE was readily absorbed after oral administration. Taken together, these results demonstrate that MPClE is a novel, potent, orally active and safe analgesic drug that targets κ-opioid receptors.

  13. Soluble expression and purification of receptor activator of nuclear factor-kappa B ligand using Escherichia coli.

    Science.gov (United States)

    Park, Sol-Ji; Lee, Se-Hoon; Kim, Kwang-Jin; Kim, Sung-Gun; Kim, Hangun; Choe, Han; Lee, Sang Yeol; Yun, Jung-Mi; Cho, Jae Youl; Chun, Jiyeon; Choi, Kap Seong; Son, Young-Jin

    2015-02-01

    Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-D-1- thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

  14. Neocortical prodynorphin expression is transiently increased with learning: Implications for time- and learning-dependent neocortical kappa opioid receptor activation.

    Science.gov (United States)

    Loh, Ryan; Collins, Sean; Galvez, Roberto

    2017-09-29

    There are several lines of evidence that indicate a prominent role for the opioid system in the acquisition and consolidation of learned associations. Specifically, kappa opioid receptor (KOR) modulation has been demonstrated to alter various behavioral tasks including whisker trace eyeblink conditioning (WTEB). WTEB is an associative conditioning paradigm in which a neutral conditioned stimulus (CS; Whisker stimulation) is paired following a short stimulus free trace interval with a salient unconditioned stimulus that elicits a blink response (US; Eye shock). Work from our laboratory has shown that WTEB conditioning is dependent upon and induces plasticity in primary somatosensory cortex (S1), a likely site for memory storage. Our subsequent studies have shown that WTEB acquisition or consolidation are impaired when the initial or later phase of KOR activation in S1 is respectively blocked. Interestingly, this mechanism by which KOR is activated in S1 during learning remains unexplored. Dynorphin (DYN), KOR's endogenous ligand, is synthesized from the precursor prodynorphin (PD) that is synthesized from preprodynorphin (PPD). In S1, most PPD is found in inhibitory GABAergic somatostatin interneurons (SOM), suggesting that these SOM interneurons are upstream regulators of learning induced KOR activation. Using immunofluorescence to investigate the expression of PD and SOM, the current study found that PD/SOM expression was transiently increased in S1 during learning. Interestingly, these findings have direct implications towards a time- and learning-dependent role for KOR activation in neocortical mechanisms mediating learning. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Salvinorin A, a kappa-opioid receptor agonist hallucinogen: pharmacology and potential template for novel pharmacotherapeutic agents in neuropsychiatric disorders

    Science.gov (United States)

    Butelman, Eduardo R.; Kreek, Mary Jeanne

    2015-01-01

    Salvinorin A is a potent hallucinogen, isolated from the ethnomedical plant Salvia divinorum. Salvinorin A is a selective high efficacy kappa-opioid receptor (KOPr) agonist, and thus implicates the KOPr system and its endogenous agonist ligands (the dynorphins) in higher functions, including cognition and perceptual effects. Salvinorin A is the only selective KOPr ligand to be widely available outside research or medical settings, and salvinorin A-containing products have undergone frequent non-medical use. KOPr/dynorphin systems in the brain are known to be powerful counter-modulatory mechanisms to dopaminergic function, which is important in mood and reward engendered by natural and chemical reinforcers (including drugs of abuse). KOPr activation (including by salvinorin A) can thus cause aversion and anhedonia in preclinical models. Salvinorin A is also a completely new scaffold for medicinal chemistry approaches, since it is a non-nitrogenous neoclerodane, unlike other known opioid ligands. Ongoing efforts have the goal of discovering novel semi-synthetic salvinorin analogs with potential KOPr-mediated pharmacotherapeutic effects (including partial agonist or biased agonist effects), with a reduced burden of undesirable effects associated with salvinorin A. PMID:26441647

  16. A unique natural selective kappa-opioid receptor agonist, salvinorin A, and its roles in human therapeutics.

    Science.gov (United States)

    Cruz, André; Domingos, Sara; Gallardo, Eugenia; Martinho, Ana

    2017-05-01

    Until the mid-60s, only the Mazatecs, an indigenous group from Oaxaca, Mexico, used Salvia Divinorum (S. divinorum) due to its hallucinogen properties. Later it was found that the hallucinogen effects of this plant were caused by the presence of a neoclerodane diterpene Salvinorin A (salvinorin A), which is a highly selective agonist of kappa-opioid receptor (KOR) that cause more intense hallucinations than the common hallucinogens as lysergic acid, mushrooms, ecstasy and others. In fact, smoking of only 200-500 μg of S. divinorum leaves is enough to produce these effects thus making it the most potent natural occurring hallucinogen known. Due to its legal status in various countries, this compound has gained a worldwide popularity as a drug of abuse with an easy access through smartshops and internet. Furthermore, salvinorin A gathered an increased interest in the scientific community thanks to its unique structure and properties, and various studies demonstrated that salvinorin A has antinociceptive, antidepressant, in some circumstances pro-depressant and anti-addictive effects that have yielded potential new avenues for research underlying salvinorin A and its semi-synthetic analogs as therapeutic agents.

  17. Differential signaling properties at the kappa opioid receptor of 12-epi-salvinorin A and its analogues.

    Science.gov (United States)

    Béguin, Cécile; Potuzak, Justin; Xu, Wei; Liu-Chen, Lee-Yuan; Streicher, John M; Groer, Chad E; Bohn, Laura M; Carlezon, William A; Cohen, Bruce M

    2012-01-15

    The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12-epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as β-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the β-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the β-arrestin mediated signaling pathway activated through KOPR.

  18. Salvinorin A, a kappa-opioid receptor agonist hallucinogen: pharmacology and potential template for novel pharmacotherapeutic agents in neuropsychiatric disorders.

    Science.gov (United States)

    Butelman, Eduardo R; Kreek, Mary Jeanne

    2015-01-01

    Salvinorin A is a potent hallucinogen, isolated from the ethnomedical plant Salvia divinorum. Salvinorin A is a selective high efficacy kappa-opioid receptor (KOPr) agonist, and thus implicates the KOPr system and its endogenous agonist ligands (the dynorphins) in higher functions, including cognition and perceptual effects. Salvinorin A is the only selective KOPr ligand to be widely available outside research or medical settings, and salvinorin A-containing products have undergone frequent non-medical use. KOPr/dynorphin systems in the brain are known to be powerful counter-modulatory mechanisms to dopaminergic function, which is important in mood and reward engendered by natural and chemical reinforcers (including drugs of abuse). KOPr activation (including by salvinorin A) can thus cause aversion and anhedonia in preclinical models. Salvinorin A is also a completely new scaffold for medicinal chemistry approaches, since it is a non-nitrogenous neoclerodane, unlike other known opioid ligands. Ongoing efforts have the goal of discovering novel semi-synthetic salvinorin analogs with potential KOPr-mediated pharmacotherapeutic effects (including partial agonist or biased agonist effects), with a reduced burden of undesirable effects associated with salvinorin A.

  19. Tramadol reduces the 5-HTP-induced head-twitch response in mice via the activation of mu and kappa opioid receptors.

    Science.gov (United States)

    Sun, Hong-Lei; Zheng, Ji-Wang; Wang, Keng; Liu, Rui-Ke; Liang, Jian-Hui

    2003-01-31

    Tramadol, an atypical opioid analgesic, stimulates both opiatergic and serotonergic systems. Here we have investigated the effect of tramadol in mice on 5-hydroxyptrytophan (5-HTP)-induced head twitch response (HTR), which is an animal model for the activation of the CNS 5-HT(2A) receptors in mice. Tramadol attenuated 5-HTP-induced HTR in a dose-dependent manner as morphine. Furthermore, the nonselective opioid receptor antagonists, naloxone and diprenorphine (M5050), reversed the effect of tramadol on 5-HTP-induced HTR dose-dependently. Interestingly, in contrast to the selective delta opioid receptor antagonist NTI, beta-FNA, a selective mu receptor antagonist, and nor-BNI, a selective kappa opioid receptor antagonist, antagonized the attenuation of 5-HTP-induced HTR by tramadol. In conclusion, administration of tramadol systemically inhibits 5-HTP-induced HTR in mice by activating opiatergic system in the CNS. Our findings show that mu and kappa opioid receptors, but not delta opioid receptor, play an important role in the regulation of serotonergic function in the CNS.

  20. Effects of ibogaine and noribogaine on the antinociceptive action of mu-, delta- and kappa-opioid receptor agonists in mice.

    Science.gov (United States)

    Bhargava, H N; Cao, Y J; Zhao, G M

    1997-03-28

    Ibogaine, an alkaloid isolated from the bark of the African shrub, Tabernanthe iboga, has been claimed to decrease the self-administration of drugs of abuse like morphine, cocaine and alcohol. To determine whether these effects are mediated via opioid receptor systems, the effects of ibogaine and its metabolite, noribogaine on the antinociceptive actions of morphine, U-50,488H and [D-Pen2,D-Pen5]enkephalin (DPDPE) which are mu- kappa- and delta-opioid receptor agonists, respectively, were determined in male Swiss-Webster mice. Administration of morphine (7 or 10 mg/kg, s.c.), U-50,488H (15 or 25 mg/kg, i.p.) or DPDPE (10 microg/mouse, i.c.v.) produced antinociception in mice as measured by the tail-flick test. Ibogaine (10, 20 or 40 mg/kg, i.p.) by itself did not alter the tail-flick latency. The same doses of ibogaine injected 10 min before the opioid drugs did not modify the antinociceptive actions of morphine, U-50,488H or DPDPE. Ibogaine administered 4 h or 24 h prior to morphine injection did not modify the antinociceptive action of the latter. A dose of 40 mg/kg (i.p.) of noribogaine enhanced the antinociceptive activity of morphine (10 mg/kg, s.c.). Similarly, the doses of 40 and 80 mg/kg of noribogaine enhanced the antinociception produced by a smaller dose of morphine (5 mg/kg, s.c.). However, antinociception induced by U-50,488H and DPDPE was not modified by noribogaine (10-40 mg/kg). It is concluded that ibogaine, which has been suggested to decrease the self-administration of cocaine and opiates like heroin in humans, does not produce such an action by interacting directly with multiple opioid receptors. However, the metabolite of ibogaine enhances the antinociception of morphine but not of U-50,488H or DPDPE. Thus, in vivo evidence has been provided for the possible interaction of ibogaine with mu-opioid receptor following its metabolism to noribogaine.

  1. The receptor tyrosine kinase FGFR4 negatively regulates NF-kappaB signaling.

    Directory of Open Access Journals (Sweden)

    Kristine A Drafahl

    Full Text Available BACKGROUND: NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs, including the Fibroblast Growth Factor Receptors (FGFRs are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit, an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling. CONCLUSIONS/SIGNIFICANCE: These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling

  2. T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20.

    Science.gov (United States)

    Coornaert, Beatrice; Baens, Mathijs; Heyninck, Karen; Bekaert, Tine; Haegman, Mira; Staal, Jens; Sun, Lijun; Chen, Zhijian J; Marynen, Peter; Beyaert, Rudi

    2008-03-01

    The paracaspase MALT1 mediates T cell antigen receptor-induced signaling to the transcription factor NF-kappaB and is indispensable for T cell activation and proliferation. Enhanced expression of MALT1 or aberrant expression of a fusion protein of the apoptosis inhibitor API2 and MALT1 has been linked to mucosa-associated lymphoid tissue lymphoma. Despite the presence of a caspase-like domain, MALT1 proteolytic activity has not yet been demonstrated. Here we show that T cell antigen receptor stimulation induced recruitment of the NF-kappaB inhibitor A20 into a complex of MALT1 and the adaptor protein Bcl-10, leading to MALT1-mediated processing of A20. API2-MALT1 expression likewise resulted in cleavage of A20. MALT1 cleaved human A20 after arginine 439 and impaired its NF-kappaB-inhibitory function. Our studies identify A20 as a substrate of MALT1 and emphasize the importance of MALT1 proteolytic activity in the 'fine tuning' of T cell antigen receptor signaling.

  3. Prognostic value of receptor activator of nuclear factor kappa-B (RANK marker in patients with breast cancer

    Directory of Open Access Journals (Sweden)

    S. I. Zabroda

    2015-01-01

    Full Text Available Despite notable progress made in studying breast cancer (BC, the mechanisms of metastases, in view of the classification into molecular subtypes, in patients with BC remain to be fully uninvestigated, in the presence of a good prognosis in particular. To study novel diagnostic and predictive markers in a new way presents current problems in the pathology of BC. This investigation deals with the expression of osteoprotegerin (OPG in the tumor cells of patients with BC. It enrolled 83 patients with locally advanced BC (T2–4N0–3M0 who had been treated in 2003 to 2010. The inclusion criterion was a histologically verified diagnosis of invasive BC. To study the level of OPG, the investigators conducted an immunohistochemical study of biopsy sections according to the standard protocol. The mean expression of receptor activator of nuclear factor kappa-B (RANK in the BC cells was 18.7 %; itsmedian was 5 % (range, 0–90 %. The patients were divided into 2 groups according to the level of RANK expression: 1 high (higher than the median; 2 low (lower than the median. The high RANK group included 39 patients; the low RANK group comprised 44 patients. Analysis of the clinical and pathological characteristics of BC patients with regard RANK expression did not show any statistically significant differences in the presence or absence of affected regional lymph nodes, T category, and Ki-67 index. The analysis of clinical and pathomorphological and immunohistochemical characteristics in patients with breast cancer, taking into consideration RANK expression level, did not show any statistically significant differences with respect to presence or absence of affected regional lymph nodes, age, T category and Ki-67 index (р > 0.05. However, it revealed the following pattern: the high expression of RANK was more common in patients positive for estrogen and progesterone receptors than in those for negative receptors (p = 0.04.

  4. Switch from excitatory to inhibitory actions of ethanol on dopamine levels after chronic exposure: Role of kappa opioid receptors.

    Science.gov (United States)

    Karkhanis, Anushree N; Huggins, Kimberly N; Rose, Jamie H; Jones, Sara R

    2016-11-01

    Acute ethanol exposure is known to stimulate the dopamine system; however, chronic exposure has been shown to downregulate the dopamine system. In rodents, chronic intermittent exposure (CIE) to ethanol also increases negative affect during withdrawal, such as, increases in anxiety- and depressive-like behavior. Moreover, CIE exposure results in increased ethanol drinking and preference during withdrawal. Previous literature documents reductions in CIE-induced anxiety-, depressive-like behaviors and ethanol intake in response to kappa opioid receptor (KOR) blockade. KORs are located on presynaptic dopamine terminals in the nucleus accumbens (NAc) and inhibit release, an effect which has been linked to negative affective behaviors. Previous reports show an upregulation in KOR function following extended CIE exposure; however it is not clear whether there is a direct link between KOR upregulation and dopamine downregulation during withdrawal from CIE. This study aimed to examine the effects of KOR modulation on dopamine responses to ethanol of behaving mice exposed to air or ethanol vapor in a repeated intermittent pattern. First, we showed that KORs have a greater response to an agonist after moderate CIE compared to air exposed mice using ex vivo fast scan cyclic voltammetry. Second, using in vivo microdialysis, we showed that, in contrast to the expected increase in extracellular levels of dopamine following an acute ethanol challenge in air exposed mice, CIE exposed mice exhibited a robust decrease in dopamine levels. Third, we showed that blockade of KORs reversed the aberrant inhibitory dopamine response to ethanol in CIE exposed mice while not affecting the air exposed mice demonstrating that inhibition of KORs "rescued" dopamine responses in CIE exposed mice. Taken together, these findings indicate that augmentation of dynorphin/KOR system activity drives the reduction in stimulated (electrical and ethanol) dopamine release in the NAc. Thus, blockade of

  5. Relative Timing Between Kappa Opioid Receptor Activation and Cocaine Determines the Impact on Reward and Dopamine Release

    Science.gov (United States)

    Chartoff, Elena H; Ebner, Shayla R; Sparrow, Angela; Potter, David; Baker, Phillip M; Ragozzino, Michael E; Roitman, Mitchell F

    2016-01-01

    Negative affective states can increase the rewarding value of drugs of abuse and promote drug taking. Chronic cocaine exposure increases levels of the neuropeptide dynorphin, an endogenous ligand at kappa opioid receptors (KOR) that suppresses dopamine release in the nucleus accumbens (NAc) and elicits negative affective states upon drug withdrawal. However, there is evidence that the effects of KOR activation on affective state are biphasic: immediate aversive effects are followed by delayed increases in reward. The impact of KOR-induced affective states on reward-related effects of cocaine over time is not known. We hypothesize that the initial aversive effects of KOR activation increase, whereas the delayed rewarding effects decrease, the net effects of cocaine on reward and dopamine release. We treated rats with cocaine at various times (15 min to 48 h) after administration of the selective KOR agonist salvinorin A (salvA). Using intracranial self-stimulation and fast scan cyclic voltammetry, we found that cocaine-induced increases in brain stimulation reward and evoked dopamine release in the NAc core were potentiated when cocaine was administered within 1 h of salvA, but attenuated when administered 24 h after salvA. Quantitative real-time PCR was used to show that KOR and prodynorphin mRNA levels were decreased in the NAc, whereas tyrosine hydroxylase and dopamine transporter mRNA levels and tissue dopamine content were increased in the ventral tegmental area 24 h post-salvA. These findings raise the possibility that KOR activation—as occurs upon withdrawal from chronic cocaine—modulates vulnerability to cocaine in a time-dependent manner. PMID:26239494

  6. Osteonecrosis of the jaw induced by receptor activator of nuclear factor-kappa B ligand (Denosumab) - Review

    Science.gov (United States)

    Brizeno, Luiz-André-Cavalcante; de Sousa, Fabrício-Bitu; Mota, Mário-Rogério-Lima; Alves, Ana-Paula-Negreiros-Nunes

    2016-01-01

    Background Denosumab, an anti-resorptive agent, IgG2 monoclonal antibody for human Receptor activator of nuclear factor-kappa B ligand (RANKL), has been related to the occurrence of osteonecrosis of the jaws. Thus, the aim of this study was to review the literature from clinical case reports, regarding the type of patient and the therapeutic approach used for osteonecrosis of the jaws induced by chronic use of Denosumab. Material and Methods For this, a literature review was performed on PubMed, Medline and Cochrane databases, using the keywords “Denosumab” “anti-RANK ligand” and “Osteonecrosis of jaw”. To be included, articles should be a report or a serie of clinical cases, describing patients aged 18 years or over who used denosumab therapy and have received any therapy for ONJ. Results Thirteen complete articles were selected for this review, totaling 17 clinical cases. The majority of ONJ cases, patients receiving Denosumab as treatment for osteoporosis and prostate cancer therapy. In most cases, patients affected by ONJ were women aged 60 or over and posterior mandible area was the main site of involvement. Diabetes pre-treatment with bisphosphonates and exodontia were the most often risk factors related to the occurrence of this condition. It is concluded that the highest number of ONJ cases caused by the use of anti-RANKL agents occurred in female patients, aged 60 years or older, under treatment for osteoporosis and cancer metastasis, and the most affected region was the mandible posterior. Conclusions The results presented in this article are valid tool supporting the non-invasive mapping of facial vascularization. Key words:Denosumab, osteonecrosis, adverse effects, osteoporosis, antineoplastic protocols. PMID:26827069

  7. Osteoprotegerin and soluble receptor activator of nuclear factor-kappa B ligand in exudative age-related macular degeneration.

    Directory of Open Access Journals (Sweden)

    Amir Ghorbanihaghjo

    2014-04-01

    Full Text Available Calcification and inflammation are among the important cases of exudative age-related macular degeneration (E-ARMD. The aim of the present study was to elucidate if there is any relationship between serum Osteoprotegerin (OPG, soluble receptor activator of nuclear factor-kappa B ligand (RANK-ligand and E-ARMD. In a cross-sectional study, we compared 45 E-ARMD patients with 45 matched controls. Diagnosis was confirmed by fluorescein angiography. Serum samples were analyzed for OPG, RANK-ligand, low density lipoprotein cholesterol (LDL-C, high density lipoprotein cholesterol (HDL-C, total cholesterol (TC, and triglyceride (TG. The levels of OPG and RANK-ligand were measured by ELISA methods. The mean age was 72.0±11.5 years in the E-ARMD group and 68.2±8.9 years in the control group (p=0.09. The level of serum OPG was 132.10±75.49 pg/ml in the E-ARMD group and 94.88±61.65 pg/ml in the control subjects. E-ARMD patients had significantly high levels of OPG (p=0.012, as well as significantly high levels of LDL-C and TC (p=0.001 and p=0.005, respectively. We could not find any significant difference in RANK-ligand, HDL-C, or TG between two study groups (p>0.05. To the best of our knowledge, this is the first study investigating the levels of OPG in E-ARMD patients. The present study showed that E-ARMD patients had high levels of serum OPG. It may act as a protective factor for E-ARMD or only as a secondary phenomenon of different processes of E-ARMD. Further prospective studies would be necessary for prognostic and predictive significance of OPG in patients affected by E-ARMD.

  8. Comparison of pharmacological activities of three distinct kappa ligands (Salvinorin A, TRK-820 and 3FLB) on kappa opioid receptors in vitro and their antipruritic and antinociceptive activities in vivo.

    Science.gov (United States)

    Wang, Yulin; Tang, Kang; Inan, Saadet; Siebert, Daniel; Holzgrabe, Ulrike; Lee, David Y W; Huang, Peng; Li, Jian-Guo; Cowan, Alan; Liu-Chen, Lee-Yuan

    2005-01-01

    Salvinorin A, TRK-820 (17-cyclopropylmethyl-3,14beta-dihydroxy-4,5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl) acrylamido]morphinan hydrochloride), and 3FLB (diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate) are structurally distinctly different from U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate], the prototypic selective kappa agonist. Here, we investigated their in vitro pharmacological activities on receptors expressed in Chinese hamster ovary cells and in vivo antiscratch and antinociceptive activities in mice. All three compounds showed high selectivity for the kappa opioid receptor (KOR) over the mu opioid receptor (MOR) and delta opioid receptor (DOR) and nociceptin or orphanin FQ receptors. In the guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, all three were full agonists on the KOR. The rank order of affinity and potency for the KOR was TRK-820 > U50,488H approximately salvinorin A > 3FLB. TRK-820 acted as a partial agonist on MOR and DOR, whereas salvinorin A and 3FLB showed no activities on these receptors. Salvinorin A, TRK-820, and 3FLB caused internalization of the human KOR in a dose-dependent manner. Interestingly, although salvinorin A and U50,488H had similar potencies in stimulating [(35)S]GTPgammaS binding, salvinorin A was about 40-fold less potent than U50,488H in promoting internalization. Following 4-h incubation, all three compounds induced down-regulation of the human KOR, with salvinorin A causing a lower extent of down-regulation. Although TRK-820 was potent and efficacious against compound 48/80-induced scratching, salvinorin A showed low and inconsistent effects, and 3FLB was inactive. In addition, salvinorin A and 3FLB were not active in the acetic acid abdominal constriction test. The discrepancy between in vitro and in vivo results may be due to in vivo metabolism of salvinorin A and 3FLB and

  9. Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Takeshita, Harunori [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Kitano, Masayasu, E-mail: mkitano6@hyo-med.ac.jp [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi [Department of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima Kobe, Hyogo 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sato, Chieri; Sekiguchi, Masahiro; Azuma, Naoto [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Miyazawa, Keiji [Discovery Research III, Research and Development, Kissei Pharmaceutical Company, 4365-1 Hodakakashiwara, Azumino, Nagano 399-8304 (Japan); Hla, Timothy [Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 1300 York Avenue, Box 69, NY 10065 (United States); Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

  10. Selective involvement of kappa opioid and phencyclidine receptors in the analgesic and motor effects of dynorphin-A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro.

    Science.gov (United States)

    Shukla, V K; Bansinath, M; Dumont, M; Lemaire, S

    1992-09-18

    Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia; 1-8 nmol) injected intracerebroventricularly in the mouse produces two independent behavioral effects: (1) a norbinaltorphimine (kappa opioid antagonist)-reversible analgesia in the acetic acid-induced writhing test and (2) motor dysfunction characterized by wild running, pop-corn jumping, hindlimb jerking and barrel rolling and antagonized by the irreversible phencyclidine (PCP) and sigma (sigma) receptor antagonist, metaphit and the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, dextromethorphan and ketamine. The specific involvement of the PCP receptor in the motor effects of Dyn Ia is supported by the direct competitive interaction of the peptide with the binding of [3H]MK-801 (Ki: 0.63 microM) and [3H]TCP (Ki: 4.6 microM) to mouse brain membrane preparations.

  11. Kinetic modeling of 11C-LY2795050, a novel antagonist radiotracer for PET imaging of the kappa opioid receptor in humans

    OpenAIRE

    Naganawa, Mika; Zheng, Ming-Qiang; Nabulsi, Nabeel; Tomasi, Giampaolo; Henry, Shannan; Lin, Shu-Fei; Ropchan, Jim; Labaree, David; Tauscher, Johannes; Neumeister, Alexander; Carson, Richard E.; Huang, Yiyun

    2014-01-01

    11C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of 11C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the bes...

  12. A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains.

    Science.gov (United States)

    Düwel, Michael; Welteke, Verena; Oeckinghaus, Andrea; Baens, Mathijs; Kloo, Bernhard; Ferch, Uta; Darnay, Bryant G; Ruland, Jürgen; Marynen, Peter; Krappmann, Daniel

    2009-06-15

    The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.

  13. TNF-alpha and 9-cis-retinoic acid synergistically induce ICAM-1 expression: evidence for interaction of retinoid receptors with NF-kappa B.

    Science.gov (United States)

    Chadwick, C C; Shaw, L J; Winneker, R C

    1998-03-15

    TNF-alpha and 9-cis-retinoic acid (9-cis-R) synergistically enhance ICAM-1 protein expression in immortalized human aortic endothelial cells (HAECTs). At a TNF-alpha concentration of 0.1 ng/ml, 1 microM 9-cis-R enhanced ICAM-1 protein expression 4-fold. Treatment with 1 microM 9-cis-R alone caused no induction of ICAM-1 expression. Functional analysis of human ICAM-1 promoter-luciferase constructs revealed that the synergism was attributable to transcriptional regulation. Expression of a luciferase reporter vector containing a 311-bp fragment of the ICAM-1 promoter (-252 to + 59 bp relative to the transcriptional start site) was increased 2.9- and 4.9-fold by treatment with 9-cis-R and TNF-alpha, respectively, while cotreatment with 9-cis-R and TNF-alpha induced expression to 19.9-fold. Mutation studies revealed that RARE and NF-kappa B sites located respectively at -226 and -188 bp relative to the transcription start site are essential for the synergistic control of promoter activity. Mutation of either the RARE or the NF-kappa B site eliminated the synergistic enhancement of promoter activity. Moreover, mutation of the RARE abrogated promoter activity induced by treatment with TNF-alpha alone and mutation of the NF-kappa B site eliminated promoter activity induced by treatment with 9-cis-R alone. We conclude that retinoid receptors and NF-kappa B act in concert at the promoter level to facilitate ICAM-1 expression in endothelial cells.

  14. A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

    Science.gov (United States)

    Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

    2016-11-04

    When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of VH and VL genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Ocean acidification weakens the immune response of blood clam through hampering the NF-kappa β and toll-like receptor pathways.

    Science.gov (United States)

    Liu, Saixi; Shi, Wei; Guo, Cheng; Zhao, Xinguo; Han, Yu; Peng, Chao; Chai, Xueliang; Liu, Guangxu

    2016-07-01

    The impact of pCO2 driven ocean acidification on marine bivalve immunity remains poorly understood. To date, this impact has only been investigated in a few bivalve species and the underlying molecular mechanism remains unknown. In the present study, the effects of the realistic future ocean pCO2 levels (pH at 8.1, 7.8, and 7.4) on the total number of haemocyte cells (THC), phagocytosis status, blood cell types composition, and expression levels of twelve genes from the NF-kappa β signaling and toll-like receptor pathways of a typical bottom burrowing bivalve, blood clam (Tegillarca granosa), were investigated. The results obtained showed that while both THC number and phagocytosis frequency were significantly reduced, the percentage of red and basophil granulocytes were significantly decreased and increased, respectively, upon exposure to elevated pCO2. In addition, exposure to pCO2 acidified seawater generally led to a significant down-regulation in the inducer and key response genes of NF-kappa β signaling and toll-like receptor pathways. The results of the present study revealed that ocean acidification may hamper immune responses of the bivalve T. granosa which subsequently render individuals more susceptible to pathogens attacks such as those from virus and bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening.

    Science.gov (United States)

    Zhang, Xiaoping; Wang, Jingxue; Wen, Kun; Mou, Zhirong; Zou, Liyun; Che, Xiaoyan; Ni, Bing; Wu, Yuzhang

    2009-12-01

    The receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays an important role in viral infection, and is a potential major neutralizing determinant. In this study, three hybridoma cell lines secreting specific monoclonal antibodies against the RBD of the S protein were generated and their exact binding sites were identified. Using yeast surface display, the binding sites of these antibodies were defined to two linear regions on the RBD: S(337-360) and S(380-399). Using these monoclonal antibodies in phage peptide library screening identified 10 distinct mimotopes 12 amino acids in length. Sequence comparison between native epitopes and these mimotopes further confirmed the binding sites, and revealed key amino acid residues involved in antibody binding. None of these antibodies could neutralize the murine leukemia virus pseudotyped expressing the SARS-CoV spike protein (MLV/SARS-CoV). However, these mAbs could be useful in the diagnosis of SARS-CoV due to their exclusive reactivity with SARS-CoV. Furthermore, this study established a feasible platform for epitope mapping. Yeast surface display combined with phage peptide library screening provides a convenient strategy for the identification of epitope peptides from certain antigenic proteins.

  17. Phage therapy pharmacology: calculating phage dosing.

    Science.gov (United States)

    Abedon, Stephen

    2011-01-01

    Phage therapy, which can be described as a phage-mediated biocontrol of bacteria (or, simply, biocontrol), is the application of bacterial viruses-also bacteriophages or phages-to reduce densities of nuisance or pathogenic bacteria. Predictive calculations for phage therapy dosing should be useful toward rational development of therapeutic as well as biocontrol products. Here, I consider the theoretical basis of a number of concepts relevant to phage dosing for phage therapy including minimum inhibitory concentration (but also "inundation threshold"), minimum bactericidal concentration (but also "clearance threshold"), decimal reduction time (D value), time until bacterial eradication, threshold bacterial density necessary to support phage population growth ("proliferation threshold"), and bacterial density supporting half-maximal phage population growth rates (K(B)). I also address the concepts of phage killing titers, multiplicity of infection, and phage peak densities. Though many of the presented ideas are not unique to this chapter, I nonetheless provide variations on derivations and resulting formulae, plus as appropriate discuss relative importance. The overriding goal is to present a variety of calculations that are useful toward phage therapy dosing so that they may be found in one location and presented in a manner that allows facile appreciation, comparison, and implementation. The importance of phage density as a key determinant of the phage potential to eradicate bacterial targets is stressed throughout the chapter.

  18. Quercetin suppresses the migration and invasion in human colon cancer Caco-2 cells through regulating toll-like receptor 4/Nuclear Factor-kappa B pathway

    Directory of Open Access Journals (Sweden)

    Mingyang Han

    2016-01-01

    Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl- 2,5-diphen yltetrazolium bromide, TLR4: Toll-like receptor 4, NF-κB: Nuclear factor-kappa B, MMP-2: Mitochondrial membrane potential-2, MMP-9: Mitochondrial membrane potential-9, TNF-α: Tumor necrosis factor-α, Cox-2: Cyclooxygenase-2, IL-6: Interleukin-6, ELISA: Enzyme-linked immunosorbent assay, PDTC: Pyrrolidine dithiocarbamate, ROS: Reactive oxygen species, DMSO: Dimethyl sulfoxide, FBS: Fetal bovine serum, DMEM: Dulbecco modified Eagle medium, OD: Optical density, IPP: Image Pro-plus, PBS: Phosphate buffered saline, SD: Standard deviation, ANOVA: One-way analysis of variance, SPSS: Statistical Package for the Social Sciences, ECM: Extracellular matrix, TLRs: Toll-like receptors, LPS: Lipopolysaccharide.

  19. Sequencing and Characterization of Pseudomonas aeruginosa phage JG004

    Directory of Open Access Journals (Sweden)

    Bunk Boyke

    2011-05-01

    Full Text Available Abstract Background Phages could be an important alternative to antibiotics, especially for treatment of multiresistant bacteria as e.g. Pseudomonas aeruginosa. For an effective use of bacteriophages as antimicrobial agents, it is important to understand phage biology but also genes of the bacterial host essential for phage infection. Results We isolated and characterized a lytic Pseudomonas aeruginosa phage, named JG004, and sequenced its genome. Phage JG004 is a lipopolysaccharide specific broad-host-range phage of the Myoviridae phage family. The genome of phage JG004 encodes twelve tRNAs and is highly related to the PAK-P1 phage genome. To investigate phage biology and phage-host interactions, we used transposon mutagenesis of the P. aeruginosa host and identified P. aeruginosa genes, which are essential for phage infection. Analysis of the respective P. aeruginosa mutants revealed several characteristics, such as host receptor and possible spermidine-dependance of phage JG004. Conclusions Whole genome sequencing of phage JG004 in combination with identification of P. aeruginosa host genes essential for infection, allowed insights into JG004 biology, revealed possible resistance mechanisms of the host bacterium such as mutations in LPS and spermidine biosynthesis and can also be used to characterize unknown gene products in P. aeruginosa.

  20. Rifampicin Inhibits the LPS-induced Expression of Toll-like Receptor 2 via the Suppression of NF-kappaB DNA-binding Activity in RAW 264.7 Cells.

    Science.gov (United States)

    Kim, Seong Keun; Kim, Young Mi; Yeum, Chung Eun; Jin, Song-Hyo; Chae, Gue Tae; Lee, Seong-Beom

    2009-12-01

    Rifampicin is a macrocyclic antibiotic which is used extensively for treatment against Mycobacterium tuberculosis and other mycobacterial infections. Recently, a number of studies have focused on the immune-regulatory effects of rifampicin. Therefore, we hypothesized that rifampicin may influence the TLR2 expression in LPS-activated RAW 264.7 cells. In this study, we determined that rifampicin suppresses LPS-induced TLR2 mRNA expression. The down-regulation of TLR2 expression coincided with decreased production of TNF-alpha. Since NF-kappaB is a major transcription factor that regulates genes for TLR2 and TNF-alpha, we examined the effect of rifampicin on the LPS-induced NF-kappaB activation. Rifampicin inhibited NF-kappaB DNA-binding activity in LPS-activated RAW 264.7 cells, while it did not affect IKKalpha/beta activity. However, rifampicin slightly inhibited the nuclear translocation of NF-kappaB p65. In addition, rifampicin increased physical interaction between pregnane X receptor, a receptor for rifampicin, and NF-kappaB p65, suggesting pregnane X receptor interferes with NF-kappaB binding to DNA. Taken together, our results demonstrate that rifampicin inhibits LPS-induced TLR2 expression, at least in part, via the suppression of NF-kappaB DNA-binding activity in RAW 264.7 cells. Thus, the present results suggest that the rifampicin-mediated inhibition of TLR2 via the suppression of NF-kappaB DNA-binding activity may be a novel mechanism of the immune-suppressive effects of rifampicin.

  1. Designing phage therapeutics.

    Science.gov (United States)

    Goodridge, Lawrence D

    2010-01-01

    Phage therapy is the application of phages to bodies, substances, or environments to effect the biocontrol of pathogenic or nuisance bacteria. To be effective, phages, minimally, must be capable of attaching to bacteria (adsorption), killing those bacteria (usually associated with phage infection), and otherwise surviving (resisting decay) until they achieve attachment and subsequent killing. While a strength of phage therapy is that phages that possess appropriate properties can be chosen from a large diversity of naturally occurring phages, a more rational approach to phage therapy also can include post-isolation manipulation of phages genetically, phenotypically, or in terms of combining different products into a single formulation. Genetic manipulation, especially in these modern times, can involve genetic engineering, though a more traditional approach involves the selection of spontaneously occurring phage mutants during serial transfer protocols. While genetic modification typically is done to give rise to phenotypic changes in phages, phage phenotype alone can also be modified in vitro, prior to phage application for therapeutic purposes, as for the sake of improving phage lethality (such as by linking phage virions to antibacterial chemicals such as chloramphenicol) or survival capabilities (e.g., via virion PEGylation). Finally, phages, both naturally occurring isolates or otherwise modified constructs, can be combined into cocktails which provide collectively enhanced capabilities such as expanded overall host range. Generally these strategies represent different routes towards improving phage therapy formulations and thereby efficacy through informed design.

  2. CoMFA analyses of C-2 position salvinorin A analogs at the kappa-opioid receptor provides insights into epimer selectivity.

    Science.gov (United States)

    McGovern, Donna L; Mosier, Philip D; Roth, Bryan L; Westkaemper, Richard B

    2010-04-01

    The highly potent and kappa-opioid (KOP) receptor-selective hallucinogen Salvinorin A and selected analogs have been analyzed using the 3D quantitative structure-affinity relationship technique Comparative Molecular Field Analysis (CoMFA) in an effort to derive a statistically significant and predictive model of salvinorin affinity at the KOP receptor and to provide additional statistical support for the validity of previously proposed structure-based interaction models. Two CoMFA models of Salvinorin A analogs substituted at the C-2 position are presented. Separate models were developed based on the radioligand used in the kappa-opioid binding assay, [(3)H]diprenorphine or [(125)I]6 beta-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([(125)I]IOXY). For each dataset, three methods of alignment were employed: a receptor-docked alignment derived from the structure-based docking algorithm GOLD, another from the ligand-based alignment algorithm FlexS, and a rigid realignment of the poses from the receptor-docked alignment. The receptor-docked alignment produced statistically superior results compared to either the FlexS alignment or the realignment in both datasets. The [(125)I]IOXY set (Model 1) and [(3)H]diprenorphine set (Model 2) gave q(2) values of 0.592 and 0.620, respectively, using the receptor-docked alignment, and both models produced similar CoMFA contour maps that reflected the stereoelectronic features of the receptor model from which they were derived. Each model gave significantly predictive CoMFA statistics (Model 1 PSET r(2)=0.833; Model 2 PSET r(2)=0.813). Based on the CoMFA contour maps, a binding mode was proposed for amine-containing Salvinorin A analogs that provides a rationale for the observation that the beta-epimers (R-configuration) of protonated amines at the C-2 position have a higher affinity than the corresponding alpha-epimers (S-configuration).

  3. Phage display reveals multiple contact sites between FhuA, an outer membrane receptor of Escherichia coli, and TonB.

    Science.gov (United States)

    Carter, David M; Gagnon, Jean-Nicolas; Damlaj, Moussab; Mandava, Suneeta; Makowski, Lee; Rodi, Diane J; Pawelek, Peter D; Coulton, James W

    2006-03-17

    The ferric hydroxamate uptake receptor FhuA from Escherichia coli transports siderophores across the outer membrane (OM). TonB-ExbB-ExbD transduces energy from the cytoplasmic membrane to the OM by contacts between TonB and OM receptors that contain the Ton box, a consensus sequence near the N terminus. Although the Ton box is a region of known contact between OM receptors and TonB, our biophysical studies established that TonB binds to FhuA through multiple regions of interaction. Panning of phage-displayed random peptide libraries (Ph.D.-12, Ph.D.-C7C) against TonB identified peptide sequences that specifically interact with TonB. Analyses of these sequences using the Receptor Ligand Contacts (RELIC) suite of programs revealed clusters of multiply aligned peptides that mapped to FhuA. These clusters localized to a continuous periplasm-accessible surface: Ton box/switch helix; cork domain/beta1 strand; and periplasmic turn 8. Guided by such matches, synthetic oligonucleotides corresponding to DNA sequences identical to fhuA were fused to malE; peptides corresponding to the above regions were displayed at the N terminus of E.coli maltose-binding protein (MBP). Purified FhuA peptides fused to MBP bound specifically to TonB by ELISA. Furthermore, they competed with ligand-loaded FhuA for binding to TonB. RELIC also identified clusters of multiply aligned peptides corresponding to the Ton box regions in BtuB, FepA, and FecA; to periplasmic turn 8 in BtuB and FecA; and to periplasmic turns 1 and 2 in FepA. These experimental outcomes identify specific molecular contacts made between TonB and OM receptors that extend beyond the well-characterized Ton box.

  4. [{sup 11}C]-GR89696, a potent kappa opiate receptor radioligand; in vivo binding of the R and S enantiomers

    Energy Technology Data Exchange (ETDEWEB)

    Ravert, Hayden T. E-mail: htr@jhu.edu; Scheffel, Ursula; Mathews, William B.; Musachio, John L.; Dannals, Robert F

    2002-01-01

    The R and S enantiomers of [{sup 11}C]GR89696, [{sup 11}C]-methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl)methyl] -1-piperazinecarboxylate, were synthesized from their appropriate chiral precursors and [{sup 11}C]methyl chloroformate. The [{sup 11}C]-labeled R enantiomer of GR89696, also known as GR103545, demonstrated high affinity in mouse brain with region to cerebellar ratios at 90 minutes of 11.4 and 8.7 for the hypothalamus and olfactory tubercle, respectively. The [{sup 11}C]-labeled S enantiomer showed low affinity and region to cerebellar ratios of 1 for all brain regions. The [{sup 11}C]-labeled GR103545 exhibited a selective and saturable binding for the kappa opioid receptor.

  5. [{sup 11}C]-methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl)methyl] -1-piperazinecarboxylate ([{sup 11}C]GR89696): synthesis and in vivo binding to kappa opiate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Ravert, Hayden T.; Mathews, William B.; Musachio, John L.; Scheffel, Ursula; Finley, Paige; Dannals, Robert F

    1999-10-01

    GR89696, racemic methyl 4-[(3,4-dichlorophenyl)acetyl]-3-[(1-pyrrolidinyl) methyl] -1-piperazinecarboxylate, a kappa opioid receptor ligand, was labeled with [{sup 11}C]methyl chloroformate. The radiochemical yield was 20% with an observed specific radioactivity of 75.5 GBq/{mu}mol at end of synthesis (2,040 mCi/{mu}mol). Five minutes after intravenous administration, 5.4% of the injected dose accumulated in mouse whole brain. Brain region to cerebellar ratios increased over time with ratios at 90 min of 7.8, 5.6, and 4.5 for the hypothalamus, olfactory tubercle, and striatum, respectively. The uptake of [{sup 11}C]GR89696 correlated with known kappa opioid receptor densities and was inhibited by kappa opioid selective drugs.

  6. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  7. Combined blockade of both mu- and kappa-opioid receptors prevents the acute orexigenic action of agouti-related protein

    NARCIS (Netherlands)

    Brugman, S; Clegg, DJ; Woods, SC; Seeley, RJ

    2002-01-01

    Agouti-related protein (AgRP) is an endogenous antagonist at the melanocortin 3 and 4 receptor in the hypothalamus. Central administration of AgRP produces a robust increase in food intake, and this effect can be blocked by administration of nonspecific opioid receptor antagonist. Such results impli

  8. Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-kappa B pathway.

    Science.gov (United States)

    Gouze, J N; Bianchi, A; Bécuwe, P; Dauça, M; Netter, P; Magdalou, J; Terlain, B; Bordji, K

    2002-01-16

    We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1 beta (IL-1 beta) [Arthritis Rheum. 44 (2001) 351-360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1 beta to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1 beta or with ROS decreased the activation of the nuclear factor kappa B, but not the activator protein-1. After treatment with IL-1 beta, glucosamine increased the expression of mRNA encoding the type II IL-1 beta receptor. These results emphasize the potential role of two regulating proteins of the IL-1 beta signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

  9. Discovery of the first small-molecule opioid pan antagonist with nanomolar affinity at mu, delta, kappa, and nociceptin opioid receptors.

    Science.gov (United States)

    Zaveri, Nurulain T; Journigan, V Blair; Polgar, Willma E

    2015-04-15

    The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [(35)S]GTPγS functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure-activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [(35)S]GTPγS assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3-6-fold more potent than those of JDTic. AT-076 is the

  10. Circulating levels of osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor-kappa B ligand, and high-sensitivity C-reactive protein in patients with active rheumatoid arthritis randomized to etanercept alone or in combination with methotrexate

    DEFF Research Database (Denmark)

    Sennels, H.; Sørensen, Steen; Østergaard, Mikkel;

    2008-01-01

    OBJECTIVE: To determine whether circulating levels of osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor-kappa B ligand (total sRANKL), and high-sensitivity C-reactive protein (hsCRP) change in patients with rheumatoid arthritis (RA) during...

  11. Engineered phages for electronics.

    Science.gov (United States)

    Cui, Yue

    2016-11-15

    Phages are traditionally widely studied in biology and chemistry. In recent years, engineered phages have attracted significant attentions for functionalization or construction of electronic devices, due to their specific binding, catalytic, nucleating or electronic properties. To apply the engineered phages in electronics, these are a number of interesting questions: how to engineer phages for electronics? How are the engineered phages characterized? How to assemble materials with engineered phages? How are the engineered phages micro or nanopatterned? What are the strategies to construct electronics devices with engineered phages? This review will highlight the early attempts to address these questions and explore the fundamental and practical aspects of engineered phages in electronics, including the approaches for selection or expression of specific peptides on phage coat proteins, characterization of engineered phages in electronics, assembly of electronic materials, patterning of engineered phages, and construction of electronic devices. It provides the methodologies and opens up ex-cit-ing op-por-tu-ni-ties for the development of a variety of new electronic materials and devices based on engineered phages for future applications.

  12. Oncogenic activation of NF-kappaB.

    Science.gov (United States)

    Staudt, Louis M

    2010-06-01

    Recent genetic evidence has established a pathogenetic role for NF-kappaB signaling in cancer. NF-kappaB signaling is engaged transiently when normal B lymphocytes respond to antigens, but lymphomas derived from these cells accumulate genetic lesions that constitutively activate NF-kappaB signaling. Many genetic aberrations in lymphomas alter CARD11, MALT1, or BCL10, which constitute a signaling complex that is intermediate between the B-cell receptor and IkappaB kinase. The activated B-cell-like subtype of diffuse large B-cell lymphoma activates NF-kappaB by a variety of mechanisms including oncogenic mutations in CARD11 and a chronic active form of B-cell receptor signaling. Normal plasma cells activate NF-kappaB in response to ligands in the bone marrow microenvironment, but their malignant counterpart, multiple myeloma, sustains a variety of genetic hits that stabilize the kinase NIK, leading to constitutive activation of the classical and alternative NF-kappaB pathways. Various oncogenic abnormalities in epithelial cancers, including mutant K-ras, engage unconventional IkappaB kinases to activate NF-kappaB. Inhibition of constitutive NF-kappaB signaling in each of these cancer types induces apoptosis, providing a rationale for the development of NF-kappaB pathway inhibitors for the treatment of cancer.

  13. DTDP-rhamnosyl transferase RfbF, is a newfound receptor-related regulatory protein for phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3.

    Science.gov (United States)

    Liang, Junrong; Li, Xu; Zha, Tao; Chen, Yuhuang; Hao, Huijing; Liu, Chang; Duan, Ran; Xiao, Yuchun; Su, Mingming; Wang, Xin; Jing, Huaiqi

    2016-03-11

    Bacteriophages and their hosts are continuously engaged in evolutionary competition. Here we isolated a lytic phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3. We firstly described the phage receptor was regulated by DTDP-rhamnosyl transferase RfbF, encoded within the rfb cluster that was responsible for the biosynthesis of the O antigens. The deletion of DTDP-rhamnosyl transferase RfbF of wild type O:3 strain caused failure in phiYe-F10 adsorption; however, the mutation strain retained agglutination with O:3 antiserum; and complementation of its mutant converted its sensitivity to phiYe-F10. Therefore, DTDP-rhamnosyl transferase RfbF was responsible for the phage infection but did not affect recognition of Y. enterocolitica O:3 antiserum. Further, the deletions in the putative O-antigen biosynthesis protein precursor and outer membrane protein had no effect on sensitivity to phiYe-F10 infection. However, adsorption of phages onto mutant HNF10-ΔO-antigen took longer time than onto the WT, suggesting that deletion of the putative O-antigen biosynthesis protein precursor reduced the infection efficiency.

  14. Phage display: concept, innovations, applications and future.

    Science.gov (United States)

    Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

    2010-01-01

    Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.

  15. Nuclear translocation of the 1,25D{sub 3}-MARRS (membrane associated rapid response to steroids) receptor protein and NF{kappa}B in differentiating NB4 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wenqing; Beilhartz, Greg; Roy, Yvette; Richard, Cynthia L.; Curtin, Maureen; Brown, Lauren; Cadieux, Danielle [Departments of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Coppolino, Marc [Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Farach-Carson, Mary C. [Department of Biological Sciences, University of Delaware, Newark, DE 19716 (United States); Nemere, Ilka [Department of Nutrition and Food Sciences, Center for Integrated BioSystems, Utah State University, Logan, UT 84322 8700 (United States); Meckling, Kelly A., E-mail: kmecklin@uoguelph.ca [Departments of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2010-04-15

    1,25 Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D{sub 3}, which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D{sub 3}-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NF{kappa}B). In unstimulated cells, 1,25D{sub 3}-MARRS can be co-immunoprecipitated with antibodies directed at NF{kappa}B, and NF{kappa}B is co-precipitated when antibodies against 1,25D{sub 3}-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D{sub 3}-MARRS and NF{kappa}B begin translocating to the nucleus within minutes of co-stimulation with 1,25D{sub 3} and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D{sub 3}-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NF{kappa}B and other factors with which it may be interacting.

  16. Hypothalamic kappa opioid receptor mediates both diet‐induced and melanin concentrating hormone–induced liver damage through inflammation and endoplasmic reticulum stress

    Science.gov (United States)

    Imbernon, Monica; Sanchez‐Rebordelo, Estrella; Romero‐Picó, Amparo; Kalló, Imre; Chee, Melissa J.; Porteiro, Begoña; Al‐Massadi, Omar; Contreras, Cristina; Fernø, Johan; Senra, Ana; Gallego, Rosalia; Folgueira, Cintia; Seoane, Luisa M.; van Gestel, Margriet; Adan, Roger A.; Liposits, Zsolt; Dieguez, Carlos; López, Miguel

    2016-01-01

    The opioid system is widely known to modulate the brain reward system and thus affect the behavior of humans and other animals, including feeding. We hypothesized that the hypothalamic opioid system might also control energy metabolism in peripheral tissues. Mice lacking the kappa opioid receptor (κOR) and adenoviral vectors overexpressing or silencing κOR were stereotaxically delivered in the lateral hypothalamic area (LHA) of rats. Vagal denervation was performed to assess its effect on liver metabolism. Endoplasmic reticulum (ER) stress was inhibited by pharmacological (tauroursodeoxycholic acid) and genetic (overexpression of the chaperone glucose‐regulated protein 78 kDa) approaches. The peripheral effects on lipid metabolism were assessed by histological techniques and western blot. We show that in the LHA κOR directly controls hepatic lipid metabolism through the parasympathetic nervous system, independent of changes in food intake and body weight. κOR colocalizes with melanin concentrating hormone receptor 1 (MCH‐R1) in the LHA, and genetic disruption of κOR reduced melanin concentrating hormone–induced liver steatosis. The functional relevance of these findings was given by the fact that silencing of κOR in the LHA attenuated both methionine choline–deficient, diet‐induced and choline‐deficient, high‐fat diet–induced ER stress, inflammation, steatohepatitis, and fibrosis, whereas overexpression of κOR in this area promoted liver steatosis. Overexpression of glucose‐regulated protein 78 kDa in the liver abolished hypothalamic κOR‐induced steatosis by reducing hepatic ER stress. Conclusions: This study reveals a novel hypothalamic–parasympathetic circuit modulating hepatic function through inflammation and ER stress independent of changes in food intake or body weight; these findings might have implications for the clinical use of opioid receptor antagonists. (Hepatology 2016;64:1086‐1104) PMID:27387967

  17. Bacteria, phages and septicemia.

    Directory of Open Access Journals (Sweden)

    Ausra Gaidelyte

    Full Text Available The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.

  18. Kinetic modeling of (11)C-LY2795050, a novel antagonist radiotracer for PET imaging of the kappa opioid receptor in humans.

    Science.gov (United States)

    Naganawa, Mika; Zheng, Ming-Qiang; Nabulsi, Nabeel; Tomasi, Giampaolo; Henry, Shannan; Lin, Shu-Fei; Ropchan, Jim; Labaree, David; Tauscher, Johannes; Neumeister, Alexander; Carson, Richard E; Huang, Yiyun

    2014-11-01

    (11)C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of (11)C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the best models to provide reliable measures of binding parameters. The rank order of (11)C-LY2795050 distribution volume (VT) matched the known regional KOR densities in the human brain. Blocking scans with naltrexone indicated no ideal reference region for (11)C-LY2795050. Three methods for calculation of the nondisplaceable distribution volume (VND) were assessed: (1) individual VND estimated from naltrexone occupancy plots, (2) mean VND across subjects, and (3) a fixed fraction of cerebellum VT. Approach (3) produced the lowest intersubject variability in the calculation of binding potentials (BPND, BPF, and BPP). Therefore, binding potentials of (11)C-LY2795050 can be determined if the specific binding fraction in the cerebellum is presumed to be unchanged by diseases and experimental conditions. In conclusion, results from the present study show the suitability of (11)C-LY2795050 to image and quantify KOR in humans.

  19. Kinetic modeling of 11C-LY2795050, a novel antagonist radiotracer for PET imaging of the kappa opioid receptor in humans

    Science.gov (United States)

    Naganawa, Mika; Zheng, Ming-Qiang; Nabulsi, Nabeel; Tomasi, Giampaolo; Henry, Shannan; Lin, Shu-Fei; Ropchan, Jim; Labaree, David; Tauscher, Johannes; Neumeister, Alexander; Carson, Richard E; Huang, Yiyun

    2014-01-01

    11C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of 11C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the best models to provide reliable measures of binding parameters. The rank order of 11C-LY2795050 distribution volume (VT) matched the known regional KOR densities in the human brain. Blocking scans with naltrexone indicated no ideal reference region for 11C-LY2795050. Three methods for calculation of the nondisplaceable distribution volume (VND) were assessed: (1) individual VND estimated from naltrexone occupancy plots, (2) mean VND across subjects, and (3) a fixed fraction of cerebellum VT. Approach (3) produced the lowest intersubject variability in the calculation of binding potentials (BPND, BPF, and BPP). Therefore, binding potentials of 11C-LY2795050 can be determined if the specific binding fraction in the cerebellum is presumed to be unchanged by diseases and experimental conditions. In conclusion, results from the present study show the suitability of 11C-LY2795050 to image and quantify KOR in humans. PMID:25182664

  20. Salvinorin A, a kappa-opioid receptor (KOP-r agonist hallucinogen: Pharmacology and potential template for novel pharmacotherapeutic agents in neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Eduardo eButelman

    2015-09-01

    Full Text Available Salvinorin A is a potent hallucinogen, isolated from the ethnomedical plant Salvia divinorum. Salvinorin A is a selective high efficacy kappa-opioid receptor (KOPr agonist, and thus implicates the KOPr system and its endogenous agonist ligands (the dynorphins in higher functions, including cognition, and perceptual effects. Salvinorin A is the only selective KOPr ligand to be widely available outside research or medical settings, and salvinorin A- containing products have undergone frequent non-medical use. KOPr/dynorphin systems in the brain are known to be powerful counter-modulatory mechanisms to dopaminergic function, which is important in mood and reward engendered by natural and drug reinforcers (including drugs of abuse. KOPr activation (including by salvinorin A can thus cause aversion and anhedonia in preclinical models. Salvinorin A is also a completely new scaffold for medicinal chemistry approaches, since it is a non-nitrogenous neoclerodane, unlike all other known opioid ligands. Ongoing efforts have the goal of discovering novel semi-synthetic salvinorin analogs with potential KOPr-mediated pharmacotherapeutic effects (including partial agonist or biased agonist effects, with a reduced burden of undesirable effects associated with salvinorin A.

  1. Effects of vitamin E on receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in rats treated with nicotine.

    Science.gov (United States)

    Norazlina, M; Maizatul-Neza, J; Azarina, A; Nazrun, A S; Norliza, M; Ima-Nirwana, S

    2010-03-01

    Vitamin E is found to reverse the effects of nicotine on bone and this study aimed to determine its mechanism. Male Sprague Dawley rats were divided into four groups and treated for 3 months: Group 1 was the control group (RC). Groups 2 (N), 3 (N+TT) and 4 (N+ATF) received nicotine 7 mg/kg throughout the treatment period. In addition, groups 3 and 4 received tocotrienol 60 mg/kg and alpha-tocopherol 60 mg/kg respectively during months 2 and 3. Parameters measured were serum osteoprotegerin (OPG), serum receptor activator of nuclear factor kappa B ligand (RANKL), femoral and lumbar bone calcium content and body weight. Nicotine did not affect OPG or RANKL levels but reduced bone calcium content suggesting the calcium loss is not due to increase osteoclastogenesis. OPG was increased in N+ATF while RANKL was slightly increased in N+TT. Both vitamin E supplements restored bone calcium loss induced by nicotine. Nicotine impaired weight gain in all treatment groups starting week 4 however, N+TT group was comparable to RC from week 6 onwards. Bone protective effects of ATF, but not TT, may be partly due to inhibition of osteoclastogenesis.

  2. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis:possible association with inflammatory cells

    Institute of Scientific and Technical Information of China (English)

    FAN Rong; SUN Bin; ZHANG Cheng-fei; L(U) Ya-lin; XUAN Wei; WANG Qian-qian; YIN Xing-zhe

    2011-01-01

    Background Receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators (RANKL and OPG) and inflammatory cell infiltration in chronic apical periodontitis.Methods The samples of chronic periapical lesions (n=40) and healthy periapical tissues (n=10) were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68.Results The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues (P<0.001). The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration (P<0.05). Significantly increased RANKL expression was found with T lymphocytes (CD3+), macrophages (CD68+) and B lymphocytes (CD20+)infiltration (P<0.05). No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration.Conclusions RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

  3. Role of liver X receptors alpha agonist on expressions of LPS-induced inflammatory response associated factor IRAK-4 and NF-kappaB in Kupffer cells

    Institute of Scientific and Technical Information of China (English)

    Wang Ding; Miao Chunmu; Gong Jianping

    2008-01-01

    Objective: To explore the role of activated liver X receptor a (LXRa) on the expressions of interleukin-1 receptor associated kinase-4 (IRAK-4) and NF-kappaB (NF-κB) in the inflammatory response which induced by LPS in the Kupffer cells and to investigate the possible mechanisms of LXRα negative regulation of inflammatory response. Methods: The Kupffer cells were isolated from male Kunming mice by collagen perfusion in situ. And these cells were divided into 4 groups: normal control group, LPS treatment group, LXRα agonist T0901317 treatment group, LPS and T0901317 combined treatment group. The LPS treatment group were treated with a final concentration of 1 μg/ml LPS in RPMI 1640 and cultured for 6 h, the T0901317 treatment group were treated with a final concentration of 5 μg/ml in RPMI 1640 and cultured for 24 h, and the combined treatment group received pre-culture for 24 h with a final concentration of 1 μg/ml T0901317 in RPMI 1640 and then cultured for 6 h with a final concentration of 5 μg/ml LPS in RPMI 1640. All groups were cultured for 30 h. The expression of LXRα, IRAK-4 and NF-κB at mRNA and protein levels were detected by real-time PCR and Western blotting, and the TNF-α and IL-1β levels were detected by ELISA. Results: The levels of LXRα mRNA and protein were highest in T0901317 group, and lowest in LPS group (P0.05). Conclusion: These date suggest that the LXR agonists can effectively up-regulate the expressions of LXRa mRNA and protein and inhibit the inflammatory response. This may be via down-regulating the expressions of IRAK4 and NF-κB at mRNA and protein levels.

  4. Prenatal exposure to vanilla or alcohol induces crawling after these odors in the neonate rat: The role of mu and kappa opioid receptor systems.

    Science.gov (United States)

    Gaztañaga, Mirari; Aranda-Fernández, P Ezequiel; Chotro, M Gabriela

    2015-09-01

    Rat fetuses can perceive chemosensory stimuli derived from their mother's diet, and they may learn about those stimuli. In previous studies we have observed that prenatal exposure to alcohol during the last days of gestation increases the acceptance and liking of an alcohol flavor in infant and adolescent rats. While these results were not found after prenatal exposure to vanilla, cineole or anise, suggesting that the pharmacological properties of alcohol, mediated by the opioid system, underlie the effects observed with this drug. Considering that other studies report enhanced acceptance of non-alcohol flavors experienced prenatally when subjects were tested before infancy, we explore the possibility of observing similar results if testing 1-day old rats exposed prenatally to vanilla. Using an "odor-induced crawling" testing procedure, it was observed that neonates exposed prenatally to vanilla or alcohol crawl for a longer distance towards the experienced odor than to other odors or than control pups. Blocking mu, but not kappa opioid receptors, reduced the attraction of vanilla odor to neonates exposed to vanilla in utero, while the response to alcohol in pups exposed prenatally to this drug was affected by both antagonists. Results confirm that exposure to a non-alcohol odor enhances postnatal responses to it, observable soon after birth, while also suggesting that the mu opioid receptor system plays an important role in generating this effect. The results also imply that with alcohol exposure, the prenatal opioid system is wholly involved, which could explain the longer retention of the enhanced attraction to alcohol following prenatal experience with the drug.

  5. Ablation of kappa-opioid receptors from brain dopamine neurons has anxiolytic-like effects and enhances cocaine-induced plasticity.

    Science.gov (United States)

    Van't Veer, Ashlee; Bechtholt, Anita J; Onvani, Sara; Potter, David; Wang, Yujun; Liu-Chen, Lee-Yuan; Schütz, Günther; Chartoff, Elena H; Rudolph, Uwe; Cohen, Bruce M; Carlezon, William A

    2013-07-01

    Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR(-/-)) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR(lox/lox)). Autoradiography demonstrated complete ablation of KOR binding in the KOR(-/-) mutants, and reduced binding in the DAT-KOR(lox/lox) mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR(-/-) mice appeared normal in the open field and light/dark box tests, DAT-KOR(lox/lox) mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR(-/-) mutants, but was exaggerated in DAT-KOR(lox/lox) mutants. Increased sensitivity to cocaine in the DAT-KOR(lox/lox) mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR(-/-) mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.

  6. Salvinorin A regulates dopamine transporter function via a kappa opioid receptor and ERK1/2-dependent mechanism.

    Science.gov (United States)

    Kivell, Bronwyn; Uzelac, Zeljko; Sundaramurthy, Santhanalakshmi; Rajamanickam, Jeyaganesh; Ewald, Amy; Chefer, Vladimir; Jaligam, Vanaja; Bolan, Elizabeth; Simonson, Bridget; Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Prisinzano, Thomas E; Gomes, Ivone; Devi, Lakshmi A; Jayanthi, Lankupalle D; Sitte, Harald H; Ramamoorthy, Sammanda; Shippenberg, Toni S

    2014-11-01

    Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP(+) accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP(+)). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signalling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Electrostatic interactions between the CTX phage minor coat protein and the bacterial host receptor TolA drive the pathogenic conversion of Vibrio cholerae.

    Science.gov (United States)

    Houot, Laetitia; Navarro, Romain; Nouailler, Matthieu; Duché, Denis; Guerlesquin, Françoise; Lloubes, Roland

    2017-08-18

    Vibrio cholerae is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTXΦ, that transmits the cholera toxin-encoding genes. This toxigenic conversion of V. cholerae has evident implication in both genome plasticity and epidemic risk, but the early stages of the infection have not been thoroughly studied. CTXΦ transit across the bacterial periplasm requires binding between the minor coat protein named pIII and a bacterial inner-membrane receptor, TolA, which is part of the conserved Tol-Pal molecular motor. To gain insight into the TolA-pIII complex, we developed a bacterial two-hybrid approach, named Oxi-BTH, suited for studying the interactions between disulfide bond-folded proteins in the bacterial cytoplasm of an Escherichia coli reporter strain. We found that two of the four disulfide bonds of pIII are required for its interaction with TolA. By combining Oxi-BTH assays, NMR, and genetic studies, we also demonstrate that two intermolecular salt bridges between TolA and pIII provide the driving forces of the complex interaction. Moreover, we show that TolA residue Arg-325 involved in one of the two salt bridges is critical for proper functioning of the Tol-Pal system. Our results imply that to prevent host evasion, CTXΦ uses an infection strategy that targets a highly conserved protein of Gram-negative bacteria essential for the fitness of V. cholerae in its natural environment. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Brovko, Luba; Orinska, Zane; Fayad, Raja; Paus, Ralf; Bulfone-Paus, Silvia

    2003-01-17

    ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate

  9. A combined ligand-based and target-based drug design approach for G-protein coupled receptors: application to salvinorin A, a selective kappa opioid receptor agonist

    Science.gov (United States)

    Singh, Nidhi; Chevé, Gwénaël; Ferguson, David M.; McCurdy, Christopher R.

    2006-08-01

    Combined ligand-based and target-based drug design approaches provide a synergistic advantage over either method individually. Therefore, we set out to develop a powerful virtual screening model to identify novel molecular scaffolds as potential leads for the human KOP (hKOP) receptor employing a combined approach. Utilizing a set of recently reported derivatives of salvinorin A, a structurally unique KOP receptor agonist, a pharmacophore model was developed that consisted of two hydrogen bond acceptor and three hydrophobic features. The model was cross-validated by randomizing the data using the CatScramble technique. Further validation was carried out using a test set that performed well in classifying active and inactive molecules correctly. Simultaneously, a bovine rhodopsin based "agonist-bound" hKOP receptor model was also generated. The model provided more accurate information about the putative binding site of salvinorin A based ligands. Several protein structure-checking programs were used to validate the model. In addition, this model was in agreement with the mutation experiments carried out on KOP receptor. The predictive ability of the model was evaluated by docking a set of known KOP receptor agonists into the active site of this model. The docked scores correlated reasonably well with experimental p K i values. It is hypothesized that the integration of these two independently generated models would enable a swift and reliable identification of new lead compounds that could reduce time and cost of hit finding within the drug discovery and development process, particularly in the case of GPCRs.

  10. Staphylococcus aureus protein A binding to osteoblast tumour necrosis factor receptor 1 results in activation of nuclear factor kappa B and release of interleukin-6 in bone infection.

    Science.gov (United States)

    Claro, Tânia; Widaa, Amro; McDonnell, Cormac; Foster, Timothy J; O'Brien, Fergal J; Kerrigan, Steven W

    2013-01-01

    Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NFκB). In turn, NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NFκB activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1-SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients.

  11. The long-term effects of stress and kappa opioid receptor activation on conditioned place aversion in male and female California mice.

    Science.gov (United States)

    Laman-Maharg, Abigail R; Copeland, Tiffany; Sanchez, Evelyn Ordoñes; Campi, Katharine L; Trainor, Brian C

    2017-08-14

    Psychosocial stress leads to the activation of kappa opioid receptors (KORs), which induce dysphoria and facilitate depression-like behaviors. However, less is known about the long-term effects of stress and KORs in females. We examined the long-term effects of social defeat stress on the aversive properties of KOR activation in male and female California mice (Peromyscus californicus) using a conditioned place aversion paradigm. Female California mice naïve to social defeat, formed a place aversion following treatment with 2.5mg/kg of the KOR agonist U50,488, but females exposed to defeat did not form a place aversion to this dose. This supports the finding by others that social defeat weakens the aversive properties of KOR agonists. In contrast, both control and stressed males formed an aversion to 10mg/kg of U50,488. We also examined EGR1 immunoreactivity, an indirect marker of neuronal activity, in the nucleus accumbens (NAc) and found that stress and treatment with 10mg/kg of U50,488 increased EGR1 immunoreactivity in the NAc core in females but reduced activation in males. The effects of stress and U50,488 on EGR1 were specific to the NAc, as we found no differences in the bed nucleus of the stria terminalis. In summary, our data indicate important sex differences in the long-term effects of stress and indicate the need for further study of the molecular mechanisms mediating the behavioral effects of KOR in both males and females. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the kappa-opioid receptor.

    Science.gov (United States)

    Yan, Feng; Bikbulatov, Ruslan V; Mocanu, Viorel; Dicheva, Nedyalka; Parker, Carol E; Wetsel, William C; Mosier, Philip D; Westkaemper, Richard B; Allen, John A; Zjawiony, Jordan K; Roth, Bryan L

    2009-07-28

    Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934-11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315(7.38) was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315(7.38). 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315(7.38) and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.

  13. An IgM-kappa rat monoclonal antibody specific for the type 1 sphingosine 1-phosphate G protein-coupled receptor with antagonist and agonist activities.

    Science.gov (United States)

    Goetzl, Edward J; Dembrow, Dale; Van Brocklyn, James R; Gráler, Markus; Huang, Mei-Chuan

    2004-04-30

    Sphingosine 1-phosphate (S1P) type 1G protein-coupled receptors (S1P1 GPCRs) are specific high-affinity transducers for this lipid growth factor and cellular mediator. S1P1 GPCRs are widely-expressed and physiologically critical in the cardiovascular and immune systems. Functional rat monoclonal antibodies (MoAbs) have been generated against human S1P1 GPCRs expressed in rat null-cell transductants to provide bioavailable agents capable of stimulating or suppressing the S1P-S1P1 GPCR axis. The rat IgM-kappa anti-S1P1 GPCR MoAb designated 4B5.2 binds specifically to native human or mouse S1P1 GPCRs in cell membranes, but not to solubilized and denatured S1P1 GPCRs. Specific binding of 32P-S1P to cellular S1P1 GPCRs is not blocked by 4B5.2. T cell chemotactic responses to S1P and S1P suppression of T cell chemotaxis to chemokines both are inhibited selectively by 4B5.2. In contrast, generation of gamma-interferon by stimulated T cells is diminished by 4B5.2 as by S1P. T cell S1P1 GPCR-selective antagonist and agonist effects of 4B5.2 in vivo may alter immune responses as distinctively as the available poly-S1P GPCR-directed pharmacological agents, without the undesirable side-effects attributable to actions of these agents on other S1P GPCRs.

  14. Divalent lead cations induce cyclooxygenase-2 gene expression by epidermal growth factor receptor/nuclear factor-kappa B signaling in A431carcinoma cells.

    Science.gov (United States)

    Chou, Yii-Her; Woon, Peng-Yeong; Huang, Wan-Chen; Shiurba, Robert; Tsai, Yao-Ting; Wang, Yu-Shiuan; Hsieh, Tusty-Jiuan; Chang, Wen-Chang; Chuang, Hung-Yi; Chang, Wei-Chiao

    2011-06-10

    Divalent lead cations (Pb²+) are toxic metal pollutants that may contribute to inflammatory diseases in people and animals. Human vascular smooth muscle cells in culture respond to low concentrations of Pb²+ ions by activating mediators of inflammation via the plasma membrane epidermal growth factor receptor (EGFR). These include cyclooxygenase-2 (COX-2) and cytosolic phospholipase A₂ as well as the hormone-like lipid compound prostaglandin E₂. To further clarify the mechanism by which Pb²+ induces such mediators of inflammation, we tested human epidermoid carcinoma cell line A431 that expresses high levels of EGFR. Reverse transcription PCR and western blots confirmed A431 cells treated with a low concentration (1 μM) of Pb²+ in the form of lead (II) nitrate increased expression of COX-2 mRNA and its encoded protein in a time-dependent manner. Promoter deletion analysis revealed the transcription factor known as nuclear factor-kappa B (NF-κB) was a necessary component of the COX-2 gene response. NF-κB inhibitor BAY 11-7082 suppressed Pb²+-induced COX-2 mRNA expression, and EGFR inhibitors AG1478 and PD153035 as well as EGFR small interfering RNA reduced the coincident nuclear translocation of NF-κB. Our findings support the hypothesis that low concentrations of Pb²+ ions incite inflammation by inducing COX-2 gene expression via the EGFR/NF-κB signal transduction pathway. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  15. Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Local Osteoporotic Canine Mandible Model for the Evaluation of Peri-Implant Bone Regeneration.

    Science.gov (United States)

    Chang, Ah Ryum; Cho, Tae Hyung; Hwang, Soon Jung

    2017-08-24

    The canine mandible is useful for studying bone regeneration after dental implant placement. However, it is limited in investigations of peri-implant osteogenesis under osteoporotic conditions due to the insignificant osteoporotic effect of ovariectomy. This study aimed at establishing a local osteoporotic model without ovariectomy by using receptor activator of nuclear factor kappa-B ligand (RANKL) in a canine mandible model. This new model was used to evaluate the effects of injectable β-tricalcium phosphate (TCP) microsphere bone grafts on peri-implant bone regeneration under osteoporotic conditions with combinations of recombinant human bone morphogenetic protein-2 (rhBMP-2). A local osteoporotic canine mandible model was designed by creating a hole in the mandibular alveolar bone, then implanting a collagen sponge soaked with 20, 40, or 60 μg RANKL into the hole, and leaving it for 2 weeks. After the establishment of the dose for maximum osteoporotic bone loss at 40 μg of RANKL, the main surgery was performed. RANKL-soaked collagen sponges were removed, and dental implants were placed with bone grafts in five groups: implant only, TCP, and TCP + rhBMP-2 at 5, 15, and 45 μg. Peri-implant bone generation was determined by radiologic and histologic evaluations at 6 weeks after dental implant placement. On performing micro-computed tomography analysis, the group with TCP + 5 μg rhBMP-2 showed the highest bone volume than the other groups and a 22% increase (p model was useful for peri-implant bone regeneration under osteoporotic conditions such as those found in geriatric patients. The injectable β-TCP bone grafts used in this study were effective in peri-implant bone generation under osteoporotic conditions, and their efficiency was enhanced at 5 μg BMP-2 compared with higher concentrations of BMP-2.

  16. Stimulation of monocytes by placental microparticles involves toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells.

    Science.gov (United States)

    Joerger-Messerli, Marianne Simone; Hoesli, Irene Mathilde; Rusterholz, Corinne; Lapaire, Olav

    2014-01-01

    Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

  17. Stimulation of monocytes by placental microparticles involves Toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells

    Directory of Open Access Journals (Sweden)

    Marianne Simone Joerger-Messerli

    2014-04-01

    Full Text Available Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggests a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro.STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR and fluorescence microscopy.STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation was blocked.Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

  18. Vibrio vulnificus phage PV94 is closely related to temperate phages of V. cholerae and other Vibrio species.

    Science.gov (United States)

    Pryshliak, Mark; Hammerl, Jens A; Reetz, Jochen; Strauch, Eckhard; Hertwig, Stefan

    2014-01-01

    Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species. In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5'-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218. We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.

  19. Vibrio vulnificus phage PV94 is closely related to temperate phages of V. cholerae and other Vibrio species.

    Directory of Open Access Journals (Sweden)

    Mark Pryshliak

    Full Text Available BACKGROUND: Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1 infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species. RESULTS: In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5'-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218. CONCLUSION: We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.

  20. Angiotensin II receptor antagonist olmesartan and NF-kappaB inhibitor as cytotoxic and apoptotic agents in MCF-7 human cell line.

    Science.gov (United States)

    Bakhtiari, Elham; Hosseini, Azar; Boroushaki, Mohammad Taher; Mousavi, Seyed Hadi

    2016-08-01

    Over expression of renin-angiotensin system (RAS) and nuclear factor-kappaB (NF-kappaB) have major role in many cancers. In this study, role of angiotensin II (Ag II) and NF-kappaB pathway in breast cancer cell line (MCF-7) proliferation were studied using olmesartan (as a novel Ag II antagonist) and Bay11-7082 (as NF-kappaB inhibitor). Cells were treated with different concentrations of olmesartan and Bay11-7082.Cell proliferation was determined by 4, 5-Dimethylthiazol-2-yl, 2, 5-diphenyl tetrazolium (MTT) assay. Apoptotic cells were evaluated using PI staining of DNA fragmentation. Olmesartan and Bay11-7082 decreased cell viability. Combination of olmesartan with Bay11-7082 also decreased cell viability as compared with single agent treatments. Results showed that apoptosis is involved in olmesartan and Bay11-7082-induced toxicity. Olmesartan and Bay11-7082 inhibit the MCF-7 cells growth indicating RAS and NF-kappaB pathway blockade lead to cytotoxicity and apoptosis induction against tumour cells. So ARBs and NF-kappaB pathway inhibitors could be considered as anticancer drugs in future.

  1. The role of NF-kappaB in endometriosis.

    Science.gov (United States)

    Kaponis, Apostolos; Iwabe, Tomio; Taniguchi, Fuminori; Ito, Masayuki; Deura, Imari; Decavalas, George; Terakawa, Naoki; Harada, Tasuku

    2012-06-01

    The nuclear factor kappaB (NF-kappaB) is a ubiquitously expressed transcription factor playing vital roles in innate immunity and other processes involving cellular survival, proliferation, and differentiation. This review highlights the importance of NF-kappaB in the pathophysiology of endometriosis. Constitutive activation of NF-kappaB has been shown in endometriotic lesions. Complex interactions of NF-kappaB with steroid receptors and apoptotic molecules in endometriosis resulting in opposing roles of NF-kappaB are discussed. NF-kappaB regulates the expression of cytokines mediating autocrine self-amplifying cycles of cytokine release and NF-kappaB activation, leading to maintenance of inflammatory reactions in endometriosis. NF-kappaB can contribute to the increased ability of endometriotic cells to invade and adhere to the peritoneal surface by regulating the expression of matrix metaloproteinases. We are presenting the role of NF-kappaB to regulate vascularization and oxidative stress in endometriotic cells. Effects of drugs used for the treatment of endometriosis on NF-kappaB pathway are presented and we show how drugs that inhibit the NF-kappaB can mediate the progression of endometriosis. Novel therapeutic strategies involving the NF-kappaB and applied in endometriosis are also discussed.

  2. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Labovsky Vivian

    2012-06-01

    Full Text Available Abstract Background While breast cancer (BC is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG, receptor activator of nuclear factor kappa B ligand (RANKL, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, stromal cell-derived factor-1 (SDF-1, and their receptors (R in 2 human BC cell lines, MDA-MB-231 and MCF-7. Methods OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses. Results MCF-7 cells released higher levels of OPG in conditioned media (CM than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. Conclusions MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

  3. Expression and localization of peroxisome proliferator-activated receptors and nuclear factor kappaB in normal and lesional psoriatic skin

    DEFF Research Database (Denmark)

    Westergaard, Majken; Henningsen, Jeanette; Johansen, Claus

    2003-01-01

    activators of PPARdelta. The expression levels of NF-kappaB p50 and p65 were not significantly altered in lesional compared with nonlesional psoriatic skin. In the basal layer of normal epidermis both p50 and p65 were sequestered in the cytoplasm, whereas p50, but not p65, localized to nuclei...... in the suprabasal layers, and this distribution was maintained in lesional psoriatic skin. In normal human keratinocytes PPAR agonists neither impaired IL-1beta-induced translocation of p65 nor IL-1beta-induced NF-kappaB DNA binding. We show that PPARdelta physically interacts with the N-terminal Rel homology......-mediated transactivation was partially relieved by forced expression of the coactivators p300 or CBP. We suggest that deficient NF-kappaB activation in chronic psoriatic plaques permitting unabated PPARdelta-mediated transactivation contributes to the pathologic phenotype of psoriasis....

  4. Phage therapy: present and future

    Science.gov (United States)

    Kolesnikova, S. G.; Tulyakova, E. N.; Moiseeva, I. Y.

    2017-01-01

    In recent years, bacteriophages are known to have become an effective alternative to antibiotic drugs. The article describes the current and potential applications of bacteriophages and phage endolysins. Also of interest is the devastating effect of phages on biofilms. The development of phage resistance is touched upon as well. Furthermore, the authors discuss the issue of laying down the rules of rational phage therapy.

  5. Estimating richness from phage metagenomes

    Science.gov (United States)

    Bacteriophages are important drivers of ecosystem functions, yet little is known about the vast majority of phages. Phage metagenomics, or the study of the collective genome of an assemblage of phages, enables the investigation of broad ecological questions in phage communities. One ecological cha...

  6. Stress-induced enhancement of ethanol intake in C57BL/6J mice with a history of chronic ethanol exposure: Involvement of kappa opioid receptors

    Directory of Open Access Journals (Sweden)

    Rachel Ivy Anderson

    2016-02-01

    Full Text Available Our laboratory has previously demonstrated that daily forced swim stress (FSS prior to ethanol drinking sessions facilitates enhanced ethanol consumption in mice with a history of chronic intermittent ethanol (CIE vapor exposure without altering ethanol intake in air-exposed controls. Because both stress and chronic ethanol exposure have been shown to activate the dynorphin/kappa opioid receptor (KOR system, the present study was designed to explore a potential role for KORs in modulating stress effects on ethanol consumption in the CIE model of dependence and relapse drinking. After stable baseline ethanol intake was established in adult male C57BL/6J mice, subjects received chronic intermittent exposure (16 hr/day x 4 days/week to ethanol vapor (CIE group or air (CTL group. Weekly cycles of inhalation exposure were alternated with 5-day limited access drinking tests (1 hour access to 15% ethanol. Experiment 1 compared effects of daily FSS and KOR activation on ethanol consumption. CIE and CTL mice were either exposed to FSS (10 min, the KOR agonist U50,488 (5 mg/kg, or a vehicle injection (non-stressed condition prior to each daily drinking session during test weeks. FSS selectively increased drinking in CIE mice. U50,488 mimicked this effect in CIE mice, but also increased drinking in CTL mice. Experiment 2 assessed effects of KOR blockade on stress-induced drinking in CIE and CTL mice. Stressed and non-stressed mice were administered the short-acting KOR antagonist LY2444296 (0 or 5 mg/kg 30 min prior to each drinking session during test weeks. FSS selectively increased ethanol consumption in CIE mice, an effect that was abolished by LY2444296 pretreatment. In Experiment 3, CIE and CTL mice were administered one of four doses of U50,488 (0,1.25, 2.5, 5.0 mg/kg one hour prior to each daily drinking test (in lieu of FSS. All doses of U50,488 increased ethanol consumption in both CIE and CTL mice. The U50,488-induced increase in drinking was

  7. Pregnane X Receptor Not Nuclear Factor-kappa B Up-regulates P-glycoprotein Expression in the Brain of Chronic Epileptic Rats Induced by Kainic Acid.

    Science.gov (United States)

    Yu, Nian; Zhang, Yan-Fang; Zhang, Kang; Cheng, Yong-Fei; Ma, Hai-Yan; Di, Qing

    2017-03-16

    Drug-resistance epilepsy (DRE) is attributed to the brain P-glycoprotein (P-gp) overexpression. We previously reported that nuclear factor-kappa B (NF-κB) played a critical role in regulating P-gp expression at the brain of the acute seizure rats. This study was extended further to investigate the interaction effect of NF-κB and pregnane X receptor (PXR) on P-gp expression at the brain of chronic epileptic rats treated with carbamazepine (CBZ). The chronic epileptic models were induced by the micro-injection of kainic acid (KA) into rats' hippocampus. Subsequently, the successful models were treated with different intervention agents of CBZ; PMA(a non-specific PXR activity inhibitor) or PDTC(a specific NF-κB activity inhibitor) respectively. The expression levels of P-gp and its encoded gene mdr1a/b were significantly up-regulated on the brain of KA-induced chronic epilepsy rats or the epilepsy rats treated with CBZ for 1 week, meanwhile with a high expression of PXR. The treatment of PMA dramatically reduced both PXR and P-gp expressions at the protein and mRNA levels in the chronic epilepsy brain. By compared to the epilepsy model group, the P-gp expression was not markedly attenuated by the inhibition of NF-κB activity with PDTC treatment, nevertheless with a decrease of NF-κB expression in this intervention group. Higher levels of proinflammatory cytokines(IL-1β, IL-6, TNF-α) were found both in the brain tissue and the serum in the epilepsy rats of each group. There was a declined trend of the pro-inflammatory cytokines expression of the PDTC treatment group but with no statistical significance. This study demonstrates for the first time that P-gp up-regulation is due to increase PXR expression in the chronic phase of epilepsy, differently from that NF-κB signaling may induce the P-gp expression in the acute seizure phase. Our results offer insights into the mechanism underlying the development of DRE using or not using CBZ treatment.

  8. Two short-acting kappa opioid receptor antagonists (zyklophin and LY2444296) exhibited different behavioral effects from the long-acting antagonist norbinaltorphimine in mouse anxiety tests.

    Science.gov (United States)

    Huang, Peng; Yakovleva, Tatyana; Aldrich, Jane V; Tunis, Julia; Parry, Christopher; Liu-Chen, Lee-Yuan

    2016-02-26

    Prototypical long-acting kappa opioid receptor (KOPR) antagonists [e.g., norbinaltorphimine (norBNI)] have been reported to exert anxiolytic-like effects in several commonly used anxiety tests in rodents including the novelty-induced hypophagia (NIH) and elevated plus maze (EPM) tests. It remains unknown if the short-acting KOPR antagonists (e.g., zyklophin and LY2444296) have similar effects. In this study effects of zyklophin and LY2444296 (s.c.) were investigated in the NIH and EPM tests in mice 1h post-injection and compared with norBNI (i.p.) 48h post-administration. In the NIH test, zyklophin at 3 and 1mg/kg, but not 0.3mg/kg, or LY2444296 at 30mg/kg decreased the latency of palatable food consumption in novel cages, but had no effect in training cages, similar to norBNI (10mg/kg). Zyklophin at 3 or 1mg/kg increased or had a trend of increasing the amount of palatable food consumption in novel cages, with no effects in training cages, further indicating its anxiolytic-like effect, but norBNI (10mg/kg) and LY2444296 (30mg/kg) did not. In the EPM test, norBNI (10mg/kg) increased open arm time and % open arm entries or time, but zyklophin at all three doses and LY2444296 (30mg/kg) had no effects. In addition, zyklophin at 3mg/kg increased numbers of close and total arm entries on EPM, suggesting increased activity; however, norBNI and LY2444296 had no effects on close and total arm entries. Thus, all three KOPR antagonists had anxiolytic-like effects in the NIH test. However, only the long-acting one (norBNI), but not the short-acting ones (zyklophin and LY2444296), demonstrated anti-anxiety like effects in the EPM test. It remains to be investigated if the differences are due to the differences in their durations of action and/or pharmacodynamic properties.

  9. Phage choice, isolation, and preparation for phage therapy.

    Science.gov (United States)

    Gill, Jason J; Hyman, Paul

    2010-01-01

    Phage therapy is the use of bacteriophages--viruses that use bacteria as their host cells--as biocontrol agents of bacteria. Currently, phage therapy is garnering renewed interest as bacterial resistance to antibiotics becomes widespread. Historically, phage therapy was largely abandoned in the West in the 1940s due to the advent of chemical antibiotics, and the unreliability of phage-based treatments when compared to antibiotics. The choice of phage strain and the methods of phage preparation are now thought to have been critical to the success or failure of phage therapy trials. Insufficiently virulent phages, especially against actual target bacteria, allow bacteria to survive treatment while poorly prepared phage stocks, even if of sufficiently virulent phages, lack the numbers of viable phages required for adequate treatment. In this review we discuss the factors that determine the methods of isolation, analysis, and identification of phage species for phage therapy. We go on to discuss the various methods available for purifying phages as well as considerations of the degree of purification which is sufficient for various applications. Lastly, we review the current practices used to prepare commercial phage therapy products.

  10. Medicinal mushroom Lingzhi or Reishi, Ganoderma lucidum (W.Curt.:Fr.) P. Karst., beta-glucan induces Toll-like receptors and fails to induce inflammatory cytokines in NF-kappaB inhibitor-treated macrophages.

    Science.gov (United States)

    Batbayar, Sainkhuu; Kim, Mi Jeong; Kim, Ha Won

    2011-01-01

    Beta-Glucan of medicinal Lingzhi or Reishi mushroom, Ganoderma lucidum (BGG), possesses immunostimulatory and anti-tumor activities. Innate immune cells are activated by the binding of beta-glucan to the dectin-1 receptor. The present study investigated the immunostimulating activities of BGG, including binding to dectin-1, secretion of cytokines and reactive oxygen species, and induction of Toll-like receptors (TLRs) in RAW264.7 mouse macrophages. Reverse transcription-polymerase chain reaction and flow cytometry were used for the cytokine and TLR analyses. A mouse inflammation antibody array was used for protein-level cytokine analysis. BGG bound to dectin-1 and induced RAW264.7 cell secretion of several cytokines, including granulocyte colony-stimulating factor, interleukin (IL)-6, regulated upon activation normal T cell expressed and secreted (RANTES), tissue inhibitor of metalloproteinase-1, and tumor necrosis factor-alpha. The secretion of these cytokines was further increased by the addition of lipopolysaccharide (LPS). BGG also induced both nitric oxide and inducible nitric oxide synthase (iNOS). Treatment with an inhibitor of nuclear factor-kappa B (NF-kappaB) reduced the induction of IL-1, IL-6, and iNOS in a concentration-dependent manner. Expressions of TLR2, TLR4, and TLR6 were increased by BGG treatment, and addition of LPS induced further induction of TLR4 and TLR6. Our result indicates that BGG induces macrophage secretion of inflammatory cytokines, which can be potentiated by the presence of LPS, likely by binding to dectin-1 and TLR-2/6 receptors, which activate NF-kappaB and prompt the secretion of cytokines.

  11. Inhibition of tumor necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB and CCAAT/enhancer-binding protein-beta.

    Science.gov (United States)

    Tian, Qingping; Miyazaki, Ryohei; Ichiki, Toshihiro; Imayama, Ikuyo; Inanaga, Keita; Ohtsubo, Hideki; Yano, Kotaro; Takeda, Kotaro; Sunagawa, Kenji

    2009-05-01

    Telmisartan, an angiotensin II type 1 receptor antagonist, was reported to be a partial agonist of peroxisome proliferator-activated receptor-gamma. Although peroxisome proliferator-activated receptor-gamma activators have been shown to have an anti-inflammatory effect, such as inhibition of cytokine production, it has not been determined whether telmisartan has such effects. We examined whether telmisartan inhibits expression of interleukin-6 (IL-6), a proinflammatory cytokine, in vascular smooth muscle cells. Telmisartan, but not valsartan, attenuated IL-6 mRNA expression induced by tumor necrosis factor-alpha (TNF-alpha). Telmisartan decreased TNF-alpha-induced IL-6 mRNA and protein expression in a dose-dependent manner. Because suppression of IL-6 mRNA expression was prevented by pretreatment with GW9662, a specific peroxisome proliferator-activated receptor-gamma antagonist, peroxisome proliferator-activated receptor-gamma may be involved in the process. Telmisartan suppressed IL-6 gene promoter activity induced by TNF-alpha. Deletion analysis suggested that the DNA segment between -150 bp and -27 bp of the IL-6 gene promoter that contains nuclear factor kappaB and CCAAT/enhancer-binding protein-beta sites was responsible for telmisartan suppression. Telmisartan attenuated TNF-alpha-induced nuclear factor kappaB- and CCAAT/enhancer-binding protein-beta-dependent gene transcription and DNA binding. Telmisartan also attenuated serum IL-6 level in TNF-alpha-infused mice and IL-6 production from rat aorta stimulated with TNF-alpha ex vivo. These data suggest that telmisartan may attenuate inflammatory process induced by TNF-alpha in addition to the blockade of angiotensin II type 1 receptor. Because both TNF-alpha and angiotensin II play important roles in atherogenesis through enhancement of vascular inflammation, telmisartan may be beneficial for treatment of not only hypertension but also vascular inflammatory change.

  12. Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-kappaB signal pathways in renal proximal tubule cells.

    Science.gov (United States)

    Lee, Yu Jin; Han, Ho Jae

    2008-03-01

    It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [(3)H]thymidine incorporation in a dose (> or =3 mg/ml)- and time (> or =3 h)-dependent manner, intracellular Ca(2+) concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca(2+) chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca(2+) chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor-kappaB (NF-kappaB) and inhibitor of NF-kappaB (IkappaB) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca(2+), PKC, EGFR, p44/42 MAPK, or NF-kappaB signal pathways blocked the BSA-induced incorporation of [(3)H]thymidine. Consequently, the inhibition of Ca(2+), PKC, EGFR, p44/42 MAPKs, or NF-kappaB blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21(WAF/Cip1) and p27(Kip1) expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca(2+)/PKC as well as the EGFR-dependent p44/42 MAPK and NF-kappaB signal pathways in PTCs.

  13. Effects of the anti-receptor activator of nuclear factor kappa B ligand denusomab on beta thalassemia major-induced osteoporosis

    Directory of Open Access Journals (Sweden)

    Mohamed A Yassin

    2014-01-01

    Full Text Available Introduction: Osteoporosis represents the second most common cause of endocrinopathy in patients with beta thalassemia major (BTM. Some drugs proved effective to reduce vertebral and non-vertebral fracture risk. Denosumab is a fully human monoclonal antibody to the receptor activator of nuclear factor kappa B ligand (RANKL, a member of the tumor necrosis factor receptor superfamily essential for osteoclastogenesis. The efficacy and safety of denosumab in BTM-induced osteoporosis has not been tested. Objective: To evaluate the efficacy and safety of anti-RANKL on the biochemical and radiological parameters of bone mineralization in patients with BTM-induced osteoporosis. Design: The study population was selected using the random sampling method from the patient′s database of our thalassemia clinic. Transfusion-dependent BTM patients above 18 years with no history of treatment with bisphosphonates were randomly selected. Bone mineral density (BMD of the lumbar spine (LS and right femoral neck (FN were measured by dual energy X-ray absorption (DEXA scan using a calibrated method. Independent factors likely to be associated with low bone mass were determined and included in the analysis to ascertain possible associations. Patients and Methods: We studied 30 patients with BTM-induced osteoporosis as per World Health Organization criteria (T Score of less than − 1.0 being defined as osteopenic and a T Score of less than − 2.5 being referred as osteoporotic. 19 males and 11 females aged between 18 and 32 years, with full pubertal development (Tanner′s stage 5 at the time of the study. Their mean serum ferritin concentration was 3557 ng ± 1488 ng/ml. Every patient underwent DEXA scan as a baseline and after 12 months of denosumab therapy. Biochemical evaluation including serum concentrations of creatinine, Na, K, calcium, phosphorus, parathormone, bone specific alkaline phosphatase and type 1 collagen carboxy telopetide (ICCT using enzyme

  14. Up-regulation of the progesterone receptor (PR)-C isoform in laboring myometrium by activation of nuclear factor-kappaB may contribute to the onset of labor through inhibition of PR function.

    Science.gov (United States)

    Condon, Jennifer C; Hardy, Daniel B; Kovaric, Kelly; Mendelson, Carole R

    2006-04-01

    Progesterone acting via the progesterone receptor (PR) plays a critical role in maintaining uterine quiescence during pregnancy. In the present study, we tested the hypothesis that the transactivating capability of the PR is down-regulated in the myometrium at term by a change in uterine PR isoform ratio resulting from local activation of the nuclear factor (NF)-kappaB pathway. Overexpression of the truncated PR-C isoform in human myometrial cells inhibited PR-B transactivation. Expression of PR isoforms, PR-A, PR-B, and PR-C, was characterized by immunoblotting and quantitative PCR (Q-PCR) in fundal and lower uterine segment myometrium from pregnant women in labor and not in labor and in the pregnant mouse uterus during late gestation. We observed a marked increase in levels of PR-C and transcriptionally active PR-B specifically in fundal myometrium of women in labor. In pregnant mouse uterus, levels of PR-B and PR-C also increased between 15 days post coitum and term, whereas expression of PR-A was dramatically up-regulated at 19 days post coitum. In studies of uterine tissues of mice injected intraamniotically with surfactant protein A and of human myometrial and T47D breast cancer cells in culture, up-regulation of PR isoform expression was observed in response to activation of the NF-kappaB pathway. Chromatin immunoprecipitation analysis revealed IL-1beta induced binding of NF-kappaB to the PR promoter. Collectively, these findings suggest that up-regulation of inhibitory PR isoform expression by NF-kappaB activation in both laboring human fundus and pregnant mouse uterus near term may inhibit PR transactivation and thereby lead to a loss of uterine quiescence and the onset of labor.

  15. Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions

    Directory of Open Access Journals (Sweden)

    Rohde Manfred

    2010-11-01

    Full Text Available Abstract Background Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF. Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages. Results We isolated and characterized a phage, named JG024, which infects a broad range of clinical and environmental P. aeruginosa strains. Sequencing of the phage genome revealed that the phage JG024 is highly related to the ubiquitous and conserved PB1-like phages. The receptor of phage JG024 was determined as lipopolysaccharide. We used an artificial sputum medium to study phage infection under conditions similar to a chronic lung infection. Alginate production was identified as a factor reducing phage infectivity. Conclusions Phage JG024 is a suitable broad host range phage which could be used in phage therapy. Phage infection experiments under simulated chronic lung infection conditions showed that alginate production reduces phage infection efficiency.

  16. Schild (apparent pA2) analysis of a kappa-opioid antagonist in Planaria.

    Science.gov (United States)

    Raffa, Robert B; Baron, David A; Tallarida, Ronald J

    2006-07-01

    Previous investigators have provided radioimmunological and immunocytochemical evidence for an enkephalinergic (opioid) system in Planaria and described naloxone-sensitive qualitative behavioral responses to kappa-opioid receptor agonists. We report the application of Schild-analysis to the antagonism of a selective kappa agonist (U-50,488H) by a selective kappa antagonist (nor-BNI) in a quantitative in vivo endpoint. The results provide further evidence of a kappa-opioid-like receptor in planarians.

  17. General, kappa, delta and mu opioid receptor antagonists mediate feeding elicited by the GABA-B agonist baclofen in the ventral tegmental area and nucleus accumbens shell in rats: reciprocal and regional interactions.

    Science.gov (United States)

    Miner, Patricia; Shimonova, Lyudmila; Khaimov, Arthur; Borukhova, Yaffa; Ilyayeva, Ester; Ranaldi, Robert; Bodnar, Richard J

    2012-03-14

    Food intake is significantly increased following administration of agonists of GABA and opioid receptors into the nucleus accumbens shell (NACs) and ventral tegmental area (VTA). GABA-A or GABA-B receptor antagonist pretreatment within the VTA or NACs differentially affects mu-opioid agonist-induced feeding elicited from the same site. Correspondingly, general or selective opioid receptor antagonist pretreatment within the VTA or NACs differentially affects GABA agonist-induced feeding elicited from the same site. Regional interactions have been evaluated in feeding studies by administering antagonists in one site prior to agonist administration in a second site. Thus, opioid antagonist-opioid agonist and GABA antagonist-GABA agonist feeding interactions have been identified between the VTA and NACs. However, pretreatment with GABA-A or GABA-B receptor antagonists in the VTA failed to affect mu opioid agonist-induced feeding elicited from the NACs, and correspondingly, these antagonists administered in the NACs failed to affect mu opioid-induced feeding elicited from the VTA. To evaluate whether regional and reciprocal VTA and NACs feeding interactions occur for opioid receptor modulation of GABA agonist-mediated feeding, the present study examined whether feeding elicited by the GABA-B agonist, baclofen microinjected into the NACs was dose-dependently blocked by pretreatment with general (naltrexone: NTX), mu (beta-funaltrexamine: BFNA), kappa (nor-binaltorphamine: NBNI) or delta (naltrindole: NTI) opioid antagonists in the VTA, and correspondingly, whether VTA baclofen-induced feeding was dose-dependently blocked by NACs pretreatment with NTX, BFNA, NBNI or NTI in rats. Bilateral pairs of cannulae aimed at the VTA and NACs were stereotaxically implanted in rats, and their food intakes were assessed following vehicle and baclofen (200 ng) in each site. Baclofen produced similar magnitudes of increased food intake following VTA and NACs treatment. Baclofen

  18. Intracerebroventricular administration of kappa-agonists induces convulsions in mice.

    Science.gov (United States)

    Bansinath, M; Ramabadran, K; Turndorf, H; Shukla, V K

    1991-07-01

    Intracerebroventricular (ICV) administration of kappa-agonists (PD 117302, U-50488H and U-69593) induced convulsions in a dose-related manner in mice. The dose at which 50% of animals convulsed (CD50) was in nmol ranges for all opioids. Among the opioids used, PD 117302 was the most potent convulsant. ICV administration of either vehicle alone or U-53445E, a non-kappa-opioid (+) enantiomer of U-50488H did not induce convulsions. The convulsive response of kappa-agonists was differentially susceptible for antagonism by naloxone and/or MR 2266. Collectively, these findings support the view that convulsions induced by kappa-agonists in mice involve stereospecific opioid receptor mechanisms. Furthermore, the convulsant effect of kappa-agonists could not be modified by pretreatment with MK-801, ketamine, muscimol or baclofen. It is concluded that kappa-opioid but not NMDA or GABA receptor mechanisms are involved in convulsions induced by kappa-agonists. These results are the first experimental evidence implicating stereospecific kappa-receptor mechanisms in opioid-induced convulsions in mice.

  19. Crossing the SJL lambda locus into kappa-knockout mice reveals a dysfunction of the lambda 1-containing immunoglobulin receptor in B cell differentiation.

    OpenAIRE

    Kim, J. Y.; Kurtz, B; Huszar, D; Storb, U

    1994-01-01

    Mice of the SJL strain produce approximately 50 times less serum lambda 1 immunoglobulin light chains than other mouse strains. The defect is genetically linked to the lambda locus, but it is unknown whether it is due to regulatory alterations or known structural changes. We find no mutation in the SJL lambda 3-1 enhancer which regulates both lambda 1 and lambda 3. To investigate the defect further, the production of lambda light chains was amplified by crossing SJL with kappa-knockout mice. ...

  20. Resveratrol-mediated SIRT-1 interactions with p300 modulate receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB signaling and inhibit osteoclastogenesis in bone-derived cells.

    Science.gov (United States)

    Shakibaei, Mehdi; Buhrmann, Constanze; Mobasheri, Ali

    2011-04-01

    Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-κB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-κB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IκBα kinase and IκBα phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-κB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-κB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-κB, inhibition of NF-κB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating

  1. Characterization of kappa 1 and kappa 2 opioid binding sites in frog (Rana esculenta) brain membrane preparation

    Energy Technology Data Exchange (ETDEWEB)

    Benyhe, S.; Varga, E.; Hepp, J.; Magyar, A.; Borsodi, A.; Wollemann, M.

    1990-09-01

    The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of (3H)ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.

  2. Transcriptional down-regulation of thromboxane A(2) receptor expression via activation of MAPK ERK1/2, p38/NF-kappaB pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2009-01-01

    BACKGROUND: We have developed an in vitro model by organ culture of rat mesenteric arteries to imitate vascular smooth muscle cell (VSMC) receptor changes in cardiovascular disease. By using this model, alteration of VSMC thromboxane A(2) (TP) receptors was studied. METHODS AND RESULTS:After organ...... abolished decreased TP receptor-mediated contraction, while inhibition of translation, cyclooxygenase or nitric oxide synthase had no effect. TP receptor mRNA stability was unchanged during organ culture. CONCLUSIONS:The present study has demonstrated for the first time that organ culture of rat mesenteric...

  3. Transcriptional Down-Regulation of Thromboxane A(2) Receptor Expression via Activation of MAPK ERK1/2, p38/NF-kappaB Pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2008-01-01

    BACKGROUND: We have developed an in vitro model by organ culture of rat mesenteric arteries to imitate vascular smooth muscle cell (VSMC) receptor changes in cardiovascular disease. By using this model, alteration of VSMC thromboxane A(2) (TP) receptors was studied. METHODS AND RESULTS:After organ...... abolished decreased TP receptor-mediated contraction, while inhibition of translation, cyclooxygenase or nitric oxide synthase had no effect. TP receptor mRNA stability was unchanged during organ culture. CONCLUSIONS:The present study has demonstrated for the first time that organ culture of rat mesenteric...

  4. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  5. A mechanism for the anti-fibrogenic effects of the pregnane X receptor (PXR) in the liver: inhibition of NF-kappaB?

    Science.gov (United States)

    Axon, A; Cowie, D E; Mann, D A; Wright, M C

    2008-04-01

    The liver is susceptible to chronic damage through exposure to a variety of toxins (e.g. alcohol) and viruses (e.g. hepatitis C). Obesity, autoimmune diseases (e.g. autoimmune hepatitis) and a variety of genetic diseases (e.g. Wilson's disease) also lead to chronic liver damage. This damage results in scarring fibrogenesis, structural disruption and functional impairment of the organ. Recent work suggests that there is cross-talk between the PXR and NF-kappaB pathways. This cross-talk may explain the observation that PXR activators inhibit liver fibrosis in in vitro and in vivo animal models of the disease. This reveiw will focus on the two transcription factors and their potential interaction.

  6. Clostridium difficile phages: still difficult?

    Directory of Open Access Journals (Sweden)

    Katherine Rose Hargreaves

    2014-04-01

    Full Text Available Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however developing suitable phages is challenging. In this review we summarise the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics.Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage-host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution.No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using whole-phages are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen.

  7. Kappa Coefficients for Circular Classifications

    NARCIS (Netherlands)

    Warrens, Matthijs J.; Pratiwi, Bunga C.

    2016-01-01

    Circular classifications are classification scales with categories that exhibit a certain periodicity. Since linear scales have endpoints, the standard weighted kappas used for linear scales are not appropriate for analyzing agreement between two circular classifications. A family of kappa coefficie

  8. Phage neutralization by sera of patients receiving phage therapy.

    Science.gov (United States)

    Łusiak-Szelachowska, Marzanna; Zaczek, Maciej; Weber-Dąbrowska, Beata; Międzybrodzki, Ryszard; Kłak, Marlena; Fortuna, Wojciech; Letkiewicz, Sławomir; Rogóż, Paweł; Szufnarowski, Krzysztof; Jończyk-Matysiak, Ewa; Owczarek, Barbara; Górski, Andrzej

    2014-08-01

    The aim of our investigation was to verify whether phage therapy (PT) can induce antiphage antibodies. The antiphage activity was determined in sera from 122 patients from the Phage Therapy Unit in Wrocław with bacterial infections before and during PT, and in sera from 30 healthy volunteers using a neutralization test. Furthermore, levels of antiphage antibodies were investigated in sera of 19 patients receiving staphylococcal phages and sera of 20 healthy volunteers using enzyme-linked immunosorbent assay. The phages were administered orally, locally, orally/locally, intrarectally, or orally/intrarectally. The rate of phage inactivation (K) estimated the level of phages' neutralization by human sera. Low K rates were found in sera of healthy volunteers (K ≤ 1.73). Low K rates were detected before PT (K ≤ 1.64). High antiphage activity of sera K > 18 was observed in 12.3% of examined patients (n = 15) treated with phages locally (n = 13) or locally/orally (n = 2) from 15 to 60 days of PT. High K rates were found in patients treated with some Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis phages. Low K rates were observed during PT in sera of patients using phages orally (K ≤ 1.04). Increased inactivation of phages by sera of patients receiving PT decreased after therapy. These results suggest that the antiphage activity in patients' sera depends on the route of phage administration and phage type. The induction of antiphage activity of sera during or after PT does not exclude a favorable result of PT.

  9. An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

    NARCIS (Netherlands)

    Ait-Azzouzene, D.; Verkoczy, L.; Peters, J.H.; Gavin, A.; Skog, P.; Vela, J.L.; Nemazee, D.

    2005-01-01

    Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly

  10. Kappa Opioids, Salvinorin A and Major Depressive Disorder.

    Science.gov (United States)

    Taylor, George T; Manzella, Francesca

    2016-01-01

    Opioids are traditionally associated with pain, analgesia and drug abuse. It is now clear, however, that the opioids are central players in mood. The implications for mood disorders, particularly clinical depression, suggest a paradigm shift from the monoamine neurotransmitters to the opioids either alone or in interaction with monoamine neurons. We have a special interest in dynorphin, the last of the major endogenous opioids to be isolated and identified. Dynorphin is derived from the Greek word for power, dynamis, which hints at the expectation that the neuropeptide held for its discoverers. Yet, dynorphin and its opioid receptor subtype, kappa, has always taken a backseat to the endogenous b-endorphin and the exogenous morphine that both bind the mu opioid receptor subtype. That may be changing as the dynorphin/ kappa system has been shown to have different, often opposite, neurophysiological and behavioral influences. This includes major depressive disorder (MDD). Here, we have undertaken a review of dynorphin/ kappa neurobiology as related to behaviors, especially MDD. Highlights include the unique features of dynorphin and kappa receptors and the special relation of a plant-based agonist of the kappa receptor salvinorin A. In addition to acting as a kappa opioid agonist, we conclude that salvinorin A has a complex pharmacologic profile, with potential additional mechanisms of action. Its unique neurophysiological effects make Salvinorina A an ideal candidate for MDD treatment research.

  11. Lipid-soluble smoke particles upregulate vascular smooth muscle ETB receptors via activation of mitogen-activating protein kinases and NF-kappaB pathways

    DEFF Research Database (Denmark)

    Xu, C.B.; Zheng, J.P.; Zhang, W.

    2008-01-01

    Cigarette smoke is a strong risk factor for cardiovascular disease. However, the underlying molecular mechanisms that lead to cigarette smoke-associated cardiovascular disease remain elusive. With functional and molecular methods, we demonstrate for the first time that lipid-soluble cigarette smoke...... particles (dimethylsulfoxide-soluble cigarette smoke particles; DSP) increased the expression of endothelin type B (ET(B)) receptors in arterial smooth muscle cells. The increased ET(B) receptors in arterial smooth muscle cells was documented as enhanced contractility (sensitive myograph technique...

  12. The role of the dynorphin-kappa opioid system in the reinforcing effects of drugs of abuse.

    Science.gov (United States)

    Wee, Sunmee; Koob, George F

    2010-06-01

    Initial hypotheses regarding the role of the kappa opioid system in drug addiction suggested that kappa receptor stimulation had anti-addictive effects. However, recent research suggests that kappa receptor antagonists may reverse motivational aspects of dependence. In the present review, we revisit the studies that measured the effects of kappa receptor ligands on the reinforcing and rewarding effects of drugs and postulate underlying neurobiological mechanisms for these effects to elaborate a more complex view of the role of kappa receptor ligands in drug addiction. The review of studies indicates that kappa receptor stimulation generally antagonizes the acute reinforcing/rewarding effects of drugs whereas kappa receptor blockade has no consistent effect. However, in a drug dependent-like state, kappa receptor blockade was effective in reducing increased drug intake. In animal models of reinstatement, kappa receptor stimulation can induce reinstatement via a stress-like mechanism. Results in conditioned place preference/aversion and intracranial self-stimulation indicate that kappa receptor agonists produce, respectively, aversive-like and dysphoric-like effects. Additionally, preclinical and postmortem studies show that administration or self-administration of cocaine, ethanol, and heroin activate the kappa opioid system. kappa receptor agonists antagonize the reinforcing/rewarding effects of drugs possibly through punishing/aversive-like effects and reinstate drug seeking through stress-like effects. Evidence suggests that abused drugs activate the kappa opioid system, which may play a key role in motivational aspects of dependence. Kappa opioid systems may have an important role in driving compulsive drug intake.

  13. Violations of the 12/23 rule at the mouse immunoglobulin kappa locus, including V kappa-V kappa rearrangement.

    Science.gov (United States)

    Vinocur, Jeffrey M; Fesnak, Andrew D; Liu, Yang; Charan, Deepshikha; Prak, Eline T Luning

    2009-07-01

    Classically, recombination between immunoglobulin gene segments uses a pair of recombination signal sequences (RSSs) with dissimilar spacers (the "12/23 rule"). Using a series of different genotyping assays, four different kinds of atypical rearrangements were identified at the murine kappa locus: (1) V kappa to V kappa, (2) J kappa to J kappa, (3) V kappa to iRS, a heptameric sequence found in the J kappa C kappa intron, and (4) a possible by-product of a rearrangement between a V kappa and the hypothetical 12-RSS side of a pre-existing signal joint. The novel V kappa-V kappa structure prompted further characterization. Sequence analysis of 14 different V kappa-V kappa rearrangements cloned from murine splenocytes and hybridomas revealed a V kappa 4 family member as one participant in 13 rearrangements, but no rearrangements contained two V kappa 4 genes. The V kappa 4 partner in the V kappa-V kappa rearrangement exhibited more trimming of nucleotides at the V kappa-V kappa junction. A signal joint derived from the inversional rearrangement of two neighboring V kappas was also recovered. These data suggest that the V kappa-V kappa structures arise via RAG-mediated, intrachromosomal recombination.

  14. Lipid-soluble smoke particles upregulate vascular smooth muscle ETB receptors via activation of mitogen-activating protein kinases and NF-kappaB pathways

    DEFF Research Database (Denmark)

    Xu, Cang-Bao; Zheng, Jian-Pu; Zhang, Wei

    2008-01-01

    ), elevated levels of ET(B) receptor mRNA (quantitative real-time PCR), and protein expressions (immunohistochemistry and Western blotting). Intracellular signaling was studied with Western blotting and phosphoELISA; this revealed that DSP induced extracellular-regulated protein kinases 1 and 2 (ERK1/2), p38...

  15. Polymorphisms in NF-kappa B, PXR, LXR, PPAR gamma and risk of inflammatory bowel disease

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Christensen, Jane; Ernst, Anja

    2011-01-01

    AIM: To investigate the contribution of polymorphisms in nuclear receptors to risk of inflammatory bowel disease (IBD). METHODS: Genotypes of nuclear factor (NF)-kappa B (NFKB1) NF kappa B -94ins/del (rs28362491); peroxisome proliferator-activated receptor (PPAR)-gamma (PPAR gamma) PPAR gamma Pro12...

  16. Killing cancer cells by targeted drug-carrying phage nanomedicines

    Directory of Open Access Journals (Sweden)

    Yacoby Iftach

    2008-04-01

    Full Text Available Abstract Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates.

  17. Development of immunoglobulin lambda-chain-positive B cells, but not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals.

    Science.gov (United States)

    Derudder, Emmanuel; Cadera, Emily J; Vahl, J Christoph; Wang, Jing; Fox, Casey J; Zha, Shan; van Loo, Geert; Pasparakis, Manolis; Schlissel, Mark S; Schmidt-Supprian, Marc; Rajewsky, Klaus

    2009-06-01

    By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

  18. Phage cocktails and the future of phage therapy.

    Science.gov (United States)

    Chan, Benjamin K; Abedon, Stephen T; Loc-Carrillo, Catherine

    2013-06-01

    Viruses of bacteria, known as bacteriophages or phages, were discovered nearly 100 years ago. Their potential as antibacterial agents was appreciated almost immediately, with the first 'phage therapy' trials predating Fleming's discovery of penicillin by approximately a decade. In this review, we consider phage therapy that can be used for treating bacterial infections in humans, domestic animals and even biocontrol in foods. Following an overview of the topic, we explore the common practice - both experimental and, in certain regions of the world, clinical - of mixing therapeutic phages into cocktails consisting of multiple virus types. We conclude with a discussion of the commercial and medical context of phage cocktails as therapeutic agents. In comparing off-the-shelf versus custom approaches, we consider the merits of a middle ground, which we deem 'modifiable'. Finally, we explore a regulatory framework for such an approach based on an influenza vaccine model.

  19. Anti-nociceptive role of neuropeptide Y in the nucleus accumbens in rats with inflammation, an effect modulated by mu- and kappa-opioid receptors

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Recent study in our laboratory showed that neuropeptide Y (NPY) plays an antinociceptive role in the nucleus accumbens (NAc) in intact rats. The present study was performed to further investigate the effect of NPY in nociceptive modulation in the NAc of rats with inflammation, and the possible interaction between NPY and the opioid systems. Experimental inflammation was induced by subcutaneous injection of carrageenan into the left hindpaw of rats. Intra-NAc administration of NPY induced a dose-dependent increase of hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations in rats with inflammation. The anti-nociceptive effect of NPY was significantly blocked by subsequent intra-NAc injection of the Y1 receptor antagonist NPY28-36, suggesting an involvement of Y1 receptor in the NPY-induced anti-nociception. Furthermore, intra-NAc administration of the opioid antagonist naloxone significantly antagonized the increased HWLs induced by preceding intra-NAc injection of NPY, suggesting an involvement of the endogenous opioid system in the NPY-induced anti-nociception in the NAc during inflammation. Moreover, the NPY-induced anti-nociception was attenuated by following intra-NAc injection of the μ-opioid antagonist β-funaltrexamine (β-FNA), and κ-opioid antagonist nor-binaltorphimine (norBNI), but not by δ-opioid antagonist naltrindole, indicating that μ- and κ-opioid receptors, not δ-opioid receptor, are involved in the NPY-induced anti-nociception in the NAc in rats with inflammation.

  20. The epic of phage therapy

    OpenAIRE

    Alain Dublanchet; Shawna Bourne

    2007-01-01

    The present report describes the presentation given by Dr Alain Dublanchet at the Stanier/Oxford Hygiene Symposium, held in Oxford, England, on November 10, 2004. Dr Dublanchet's lecture, entitled ‘The epic of phage therapy’, provided a sequential account of the use of phage as an antimicrobial from its discovery to its rise and fall and current rediscovery.

  1. Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-Opioid Receptor

    Science.gov (United States)

    Polepally, Prabhakar R.; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D.; Roth, Bryan L.; Zjawiony, Jordan K.

    2014-01-01

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR. PMID:25193297

  2. Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor.

    Science.gov (United States)

    Polepally, Prabhakar R; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D; Roth, Bryan L; Zjawiony, Jordan K

    2014-10-06

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure-activity relationships, and previous salvinorin A-KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR.

  3. Receptor diversity and host interaction of bacteriophages infecting Salmonella enterica serovar Typhimurium.

    Directory of Open Access Journals (Sweden)

    Hakdong Shin

    Full Text Available BACKGROUND: Salmonella enterica subspecies enterica serovar Typhimurium is a gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages, vitamin B(12 uptake outer membrane protein, BtuB (7 phages and lipopolysaccharide-related O-antigen (7 phages. TEM observation revealed that the phages using flagella (group F or BtuB (group B as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L belong to Podoviridae family. Interestingly, while some of group F phages (F-I target FliC host receptor, others (F-II target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain. CONCLUSIONS/SIGNIFICANCE: In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella.

  4. Purinergic signaling is required for fluid shear stress-induced NF-{kappa}B translocation in osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Genetos, Damian C., E-mail: dgenetos@ucdavis.edu [Department of Anatomy, Cell Biology, and Physiology, School of Veterinary Medicine, University of California, Davis, CA (United States); Karin, Norman J. [Cell Biology and Biochemistry, Pacific Northwest National Laboratory, Richland, WA (United States); Geist, Derik J. [Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN (United States); Donahue, Henry J. [Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Pennsylvania State College of Medicine, Hershey, PA (United States); Duncan, Randall L. [Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN (United States)

    2011-04-01

    Fluid shear stress regulates gene expression in osteoblasts, in part by activation of the transcription factor NF-{kappa}B. We examined whether this process was under the control of purinoceptor activation. MC3T3-E1 osteoblasts under static conditions expressed the NF-{kappa}B inhibitory protein I{kappa}B{alpha} and exhibited cytosolic localization of NF-{kappa}B. Under fluid shear stress, I{kappa}B{alpha} levels decreased, and concomitant nuclear localization of NF-{kappa}B was observed. Cells exposed to fluid shear stress in ATP-depleted medium exhibited no significant reduction in I{kappa}B{alpha}, and NF-{kappa}B remained within the cytosol. Similar results were found using oxidized ATP or Brilliant Blue G, P2X{sub 7} receptor antagonists, indicating that the P2X{sub 7} receptor is responsible for fluid shear-stress-induced I{kappa}B{alpha} degradation and nuclear accumulation of NF-{kappa}B. Pharmacologic blockage of the P2Y6 receptor also prevented shear-induced I{kappa}B{alpha} degradation. These phenomena involved neither ERK1/2 signaling nor autocrine activation by P2X{sub 7}-generated lysophosphatidic acid. Our results suggest that fluid shear stress regulates NF-{kappa}B activity through the P2Y{sub 6} and P2X{sub 7} receptor.

  5. Phages targeting infected tissues: novel approach to phage therapy.

    Science.gov (United States)

    Górski, Andrzej; Dąbrowska, Krystyna; Hodyra-Stefaniak, Katarzyna; Borysowski, Jan; Międzybrodzki, Ryszard; Weber-Dąbrowska, Beata

    2015-01-01

    While the true efficacy of phage therapy still requires formal confirmation in clinical trials, it continues to offer realistic potential treatment in patients in whom antibiotics have failed. Novel developments and approaches are therefore needed to ascertain that future clinical trials would evaluate the therapy in its optimal form thus allowing for reliable conclusions regarding the true value of phage therapy. In this article, we present our vision to develop and establish a bank of phages specific to most threatening pathogens and armed with homing peptides enabling their localization in infected tissues in densities assuring efficient and stable eradication of infection.

  6. A catalytically inactive form of protein kinase C-associated kinase/receptor interacting protein 4, a protein kinase C beta-associated kinase that mediates NF-kappa B activation, interferes with early B cell development.

    Science.gov (United States)

    Cariappa, Annaiah; Chen, Luojing; Haider, Khaleda; Tang, Mei; Nebelitskiy, Eugene; Moran, Stewart T; Pillai, Shiv

    2003-08-15

    Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) beta-associated kinase that links PKC to NF-kappaB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCbeta, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.

  7. Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo.

    Science.gov (United States)

    Baek, Jong Min; Kim, Ju-Young; Ahn, Sung-Jun; Cheon, Yoon-Hee; Yang, Miyoung; Oh, Jaemin; Choi, Min Kyu

    2016-03-01

    Dendrobium moniliforme (DM) is a well-known plant-derived extract that is widely used in Oriental medicine. DM and its chemical constituents have been reported to have a variety of pharmacological effects, including anti-oxidative, anti-inflammatory, and anti-tumor activities; however, no reports discuss the beneficial effects of DM on bone diseases such as osteoporosis. Thus, we investigated the relationship between DM and osteoclasts, cells that function in bone resorption. We found that DM significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation; DM directly induced the down-regulation of c-Fos and nuclear factor of activated T cells c1 (NFATc1) without affecting other RANKL-dependent transduction pathways. In the later stages of osteoclast maturation, DM negatively regulated the organization of filamentous actin (F-actin), resulting in impaired bone-resorbing activity by the mature osteoclasts. In addition, micro-computed tomography (μ-CT) analysis of the murine model revealed that DM had a beneficial effect on lipopolysaccharide (LPS)-mediated bone erosion. Histological analysis showed that DM attenuated the degradation of trabecular bone matrix and formation of TRAP-positive osteoclasts in bone tissues. These results suggest that DM is a potential candidate for the treatment of metabolic bone disorders such as osteoporosis.

  8. Phage display of peptide / major histocompatibility class I complexes

    DEFF Research Database (Denmark)

    Vest Hansen, N; Ostergaard Pedersen, L; Stryhn, A;

    2001-01-01

    Major histocompatibility complex class I (MHC-I) molecules sample peptides from the intracellular environment and present them to cytotoxic T cells (CTL). To establish a selection system, and, thereby, enable a library approach to identify the specificities involved (that of the MHC-I for peptides...... and subsequently that ot the T cell receptor for peptide-MHC-I complex), we have fused a single chain peptide-MHC-I complex to the phage minor coat protein, gpIII, and displayed it on filamentous phage. Expression of peptide-MHC-I complexes was shown with relevant conformation-specific monoclonal antibodies and...

  9. Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells

    Directory of Open Access Journals (Sweden)

    Chantong Boonrat

    2012-11-01

    Full Text Available Abstract Background Microglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR and glucocorticoid receptors (GR. Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB pathway in murine BV-2 microglial cells were studied. Methods BV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1 was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6, tumor necrosis factor receptor 2 (TNFR2, and 11β-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays. Results GR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11β-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF

  10. Toll-Like Receptor 4–Mediated Nuclear Factor Kappa B Activation Is Essential for Sensing Exogenous Oxidants to Propagate and Maintain Oxidative/Nitrosative Cellular Stress

    Science.gov (United States)

    Karki, Rajendra; Igwe, Orisa J.

    2013-01-01

    The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments. PMID:24058497

  11. Toll-like receptor 4-mediated nuclear factor kappa B activation is essential for sensing exogenous oxidants to propagate and maintain oxidative/nitrosative cellular stress.

    Science.gov (United States)

    Karki, Rajendra; Igwe, Orisa J

    2013-01-01

    The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.

  12. A hepatoprotective Lindera obtusiloba extract suppresses growth and attenuates insulin like growth factor-1 receptor signaling and NF-kappaB activity in human liver cancer cell lines

    Directory of Open Access Journals (Sweden)

    Stroh Thorsten

    2011-05-01

    Full Text Available Abstract Background In traditional Chinese and Korean medicine, an aqueous extract derived from wood and bark of the Japanese spice bush Lindera obtusiloba (L.obtusiloba is applied to treat inflammations and chronic liver diseases including hepatocellular carcinoma. We previously demonstrated anti-fibrotic effects of L.obtusiloba extract in hepatic stellate cells. Thus, we here consequently examine anti-neoplastic effects of L.obtusiloba extract on human hepatocellular carcinoma (HCC cell lines and the signaling pathways involved. Methods Four human HCC cell lines representing diverse stages of differentiation were treated with L.obtusiloba extract, standardized according to its known suppressive effects on proliferation and TGF-β-expression. Beside measurement of proliferation, invasion and apoptosis, effects on signal transduction and NF-κB-activity were determined. Results L.obtusiloba extract inhibited proliferation and induced apoptosis in all HCC cell lines and provoked a reduced basal and IGF-1-induced activation of the IGF-1R signaling cascade and a reduced transcriptional NF-κB-activity, particularly in the poorly differentiated SK-Hep1 cells. Pointing to anti-angiogenic effects, L.obtusiloba extract attenuated the basal and IGF-1-induced expression of hypoxia inducible factor-1α, vascular endothelial growth factor, peroxisome proliferator-activated receptor-γ, cyclooxygenase-2 and inducible nitric oxide synthase. Conclusions The traditional application of the extract is confirmed by our experimental data. Due to its potential to inhibit critical receptor tyrosine kinases involved in HCC progression via the IGF-1 signaling pathway and NF-κB, the standardized L.obtusiloba extract should be further analysed for its active compounds and explored as (complementary treatment option for HCC.

  13. Information Phage Therapy Research Should Report

    OpenAIRE

    Stephen T. Abedon

    2017-01-01

    Bacteriophages, or phages, are viruses which infect bacteria. A large subset of phages infect bactericidally and, consequently, for nearly one hundred years have been employed as antibacterial agents both within and outside of medicine. Clinically these applications are described as phage or bacteriophage therapy. Alternatively, and especially in the treatment of environments, this practice instead may be described as a phage-mediated biocontrol of bacteria. Though the history of phage therap...

  14. Distinct Effects of Nalmefene on Dopamine Uptake Rates and Kappa Opioid Receptor Activity in the Nucleus Accumbens Following Chronic Intermittent Ethanol Exposure

    Directory of Open Access Journals (Sweden)

    Jamie H. Rose

    2016-07-01

    Full Text Available The development of pharmacotherapeutics that reduce relapse to alcohol drinking in patients with alcohol dependence is of considerable research interest. Preclinical data support a role for nucleus accumbens (NAc κ opioid receptors (KOR in chronic intermittent ethanol (CIE exposure-induced increases in ethanol intake. Nalmefene, a high-affinity KOR partial agonist, reduces drinking in at-risk patients and relapse drinking in rodents, potentially due to its effects on NAc KORs. However, the effects of nalmefene on accumbal dopamine transmission and KOR function are poorly understood. We investigated the effects of nalmefene on dopamine transmission and KORs using fast scan cyclic voltammetry in NAc brain slices from male C57BL/6J mice following five weeks of CIE or air exposure. Nalmefene concentration-dependently reduced dopamine release similarly in air and CIE groups, suggesting that dynorphin tone may not be present in brain slices. Further, nalmefene attenuated dopamine uptake rates to a greater extent in brain slices from CIE-exposed mice, suggesting that dopamine transporter-KOR interactions may be fundamentally altered following CIE. Additionally, nalmefene reversed the dopamine-decreasing effects of a maximal concentration of a KOR agonist selectively in brain slices of CIE-exposed mice. It is possible that nalmefene may attenuate withdrawal-induced increases in ethanol consumption by modulation of dopamine transmission through KORs.

  15. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages

    Science.gov (United States)

    Kuo, Chun-Liang; Kao, Chia-Tze; Fang, Hsin-Yuan; Huang, Tsui-Hsien; Chen, Yi-Wen; Shie, Ming-You

    2015-03-01

    Macrophage cells are the important effector cells in the immune reaction which are indispensable for osteoclastogenesis; their heterogeneity and plasticity renders macrophages a primer target for immune system modulation. In recent years, there have been very few studies about the effects of macrophage cells on laser treatment-regulated osteoclastogenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL to regulate osteoclastogenesis. We used a CO2 laser as a model biostimulation to investigate the role of osteoclastogenic. We also evaluated cell viability, cell death and cathepsin K expression. The CO2 laser inhibited a receptor activator of the NF-ĸB ligand (RANKL)-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that irradiation for two times reduced RANKL-enhanced TRAP activity in a dose-dependent manner. Furthermore, CO2 laser-treatment diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and NF-ĸB activation. The current report demonstrates that CO2 laser abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts and the maturation of osteoclasts. Therefore, the current results serve as an improved explanation of the cellular roles of macrophage cell populations in osteoclastogenesis as well as in alveolar bone remodeling by CO2 laser-treatment.

  16. A gut feeling of the PXR, PPAR and NF-kappaB connection.

    Science.gov (United States)

    Wahli, W

    2008-06-01

    Bowel diseases reveal the complex interplay of sensing and signalling pathways in maintaining healthy homeostasis of the intestine. Recent studies of the xenobiotic nuclear receptor, pregnane X receptor and the inflammatory mediator nuclear transcription factor kappaB (NF-kappaB) reveal a functional link between xenobiotic neutralization and inflammation and explain how certain xenobiotics can affect the immune response. Furthermore, another nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma) has been shown to produce beneficial effects in experimental inflammatory bowel diseases by repression of NF-kappaB thereby reducing inflammation, whilst its close relative PPAR beta/delta appears at a central position in signalling pathways involved in the progression of colon cancer. Recently accumulated knowledge on the action of these nuclear receptors and NF-kappaB in intestinal homeostasis may provide the rationale for the development of innovative treatment strategies with selective receptor modulators.

  17. Interleukin-1/toll-like receptor-induced nuclear factor kappa B signaling participates in intima hyperplasia after carotid artery balloon injury in goto-kakizaki rats: a potential target therapy pathway.

    Directory of Open Access Journals (Sweden)

    Xiaotian Zhang

    Full Text Available The value of restenosis after percutaneous coronary intervention (PCI is recognized worldwide, especially for diabetic patients. Interleukin-1/Toll-like receptor (IL-1/TLR signaling is involved in innate and adaptive immune responses, but whether and how the IL-1/TLR-induced nuclear factor kappa B (NFκB pathway plays key roles in intimal formation is unclear. The underlying mechanism of intima hyperplasia was investigated with a model of carotid balloon injury in Goto-Kakizaki (GK and Wistar rats and with lipopolysaccharide-stimulated macrophages. Elastic-van Gieson staining showed the medial area peakedon Day 3 post-injury and decreased by Day 7 post-injury in both GK and Wistar rats. The N/M at Day 7 in GK rats was significantly higher than in Wistar rats (p<0.001. The percent of 5-ethynyl-2'-deoxyuridine (EdU staining-positive cells on Day 3 post-injury was greater than seen on Day 7 post-injury in GK and Wistar rats. The percent of EdU-positive cells on Days 3 and 7 post-injury in Wistar rats was less than that found in GK rats (p<0.01; p<0.05. NFκBp65 immunostaining had increased by Day 7 post-injury. Agilent Whole Genome Oligo Microarray verified that the IL-1/TLR-induced NFκB pathway was activated by carotid balloon injury. TLR4, IL-1 receptor associated kinase, inhibitors α of NFκB, human antigen R, c-Myc (Proto-Oncogene Proteins, EGF-like module-containing mucin-like hormone receptor-like 1 and Interleukin-6 were up-regulated or down-regulated according to immunochemistry, quantitative real-time PCR, Western blotting and Enzyme linked immunosorbent assay. Overall, we conclude that the IL-1/TLR-induced NFκB pathway participates in the intimal hyperplasia after carotid injury in GK and Wistar rats and that GK rats respond more intensely to the inflammation than Wistar rats.

  18. Human Volunteers Receiving Escherichia coli Phage T4 Orally: a Safety Test of Phage Therapy

    OpenAIRE

    Bruttin, Anne; Brüssow, Harald

    2005-01-01

    Fifteen healthy adult volunteers received in their drinking water a lower Escherichia coli phage T4 dose (103 PFU/ml), a higher phage dose (105 PFU/ml), and placebo. Fecal coliphage was detected in a dose-dependent way in volunteers orally exposed to phage. All volunteers receiving the higher phage dose showed fecal phage 1 day after exposure; this prevalence was only 50% in subjects receiving the lower phage dose. No fecal phage was detectable a week after a 2-day course of oral phage applic...

  19. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    DEFF Research Database (Denmark)

    Jiang, Y P; Wang, H; D'Eustachio, P

    1993-01-01

    with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic...... indicates that expression of R-PTP-kappa in the central nervous system is developmentally regulated, with highest expression seen in actively developing areas and, in the adult, in areas capable of developmental plasticity such as the hippocampal formation and cerebral cortex. The mouse R-PTP-kappa gene...

  20. Safrole oxide induces human umbilical vein endothelial cell transdifferentiation to 5-hydroxytryptaminergic neuron-like cells through tropomyosin receptor kinase A/cyclooxygenase 2/nuclear factor-kappa B/interleukin 8 signaling.

    Science.gov (United States)

    Su, Le; Zhao, Jing; Zhao, Bao Xiang; Zhang, Shang Li; Miao, Jun Ying

    2011-10-01

    The phenomenon of endothelial-neural transdifferentiation has been observed for a long time, but the mechanism is not clear. We previously found that safrole oxide induced human umbilical vein endothelial cell transdifferentiation into neuron-like cells. In this study, we first validated that these cells induced by safrole oxide were functional 5-hydroxytryptaminergic neuron-like cells. Then, we performed microarray analysis of safrole oxide-treated and -untreated human umbilical vein endothelial cells. Safrole oxide elevated the levels of cyclooxygenase 2 (COX-2), interleukin-8 (IL-8) and reactive oxygen species (ROS), which was accompanied by nuclear factor-kappa B (NF-κB) nuclear translocation during the transdifferentiation. Blockade of tropomyosin receptor kinase A (TrkA) by an inhibitor or short hairpin RNA inhibited the levels of COX-2/IL-8 and the nuclear translocation of NF-κB but did not suppress the increased ROS level. As a result, cells underwent apoptosis. Therefore, via TrkA, safrole oxide may induce endothelial cell transdifferentiation into functional neuron-like cells. During this process, the increased levels of COX-2/IL-8 and the subsequent elevation of ROS production induced NF-κB nuclear translocation and IL-8 secretion. With the activity of TrkA inhibited, the inactive NF-κB regulated the ROS level in a negative feedback manner. Finally, the transdifferentiation pathway was blocked and cells became apoptotic. The TrkA/COX-2/IL-8 signal pathway may have an important role in endothelial-neural transdifferentiation, and safrole oxide may trigger this process by activating TrkA.

  1. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    Science.gov (United States)

    Zhang, Ailiang; Wang, Kun; Ding, Lianghua; Bao, Xinnan; Wang, Xuan; Qiu, Xubin; Liu, Jinbo

    2017-01-01

    Lumbar disc herniation (LDH) is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB) and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP) was implanted in the left L5 dorsal root ganglion (DRG) to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC), caspase-1, interleukin (IL)-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP) were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65) and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation.

  2. Expression of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) in ameloblastomas.

    Science.gov (United States)

    Kumamoto, Hiroyuki; Ooya, Kiyoshi

    2004-01-01

    To clarify the roles of osteoclast regulatory factors in progression of odontogenic tumors, expression of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL), and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) were analyzed in ameloblastomas as well as tooth germs. Tissue specimens of nine tooth germs and 36 benign and one malignant ameloblastomas were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry for the expression of PTHrP, ODF/RANKL, and OCIF/OPG. Expression of PTHrP, ODF/RANKL, and OCIF/OPG mRNA was detected in all tooth germ and ameloblastoma samples. Immunohistochemical reactivity for PTHrP was recognized in both normal and neoplastic odontogenic epithelial cells. In ameloblastomas, PTHrP reactivity in peripheral columnar or cuboidal cells was stronger than that in central polyhedral cells, and keratinizing cells showed increased PTHrP reactivity. ODF/RANKL and OCIF/OPG were expressed predominantly in mesenchymal cells rather than in odontogenic epithelial cells in both tooth germs and ameloblastomas. Epithelial ODF/RANKL and OCIF/OPG expression was slightly lower in ameloblastomas than in tooth germs. Tumor cells in plexiform ameloblastomas showed slightly higher reactivity for PTHrP and ODF/RANKL than tumor cells in follicular ameloblastomas. Expression of PTHrP, ODF/RANKL and OCIF/OPG in tooth germs and ameloblastomas suggests that these factors might locally regulate bone metabolism and dynamics in tooth development as well as in progression of ameloblastomas. These factors might also be involved in tumor cell differentiation and/or tumor tissue structuring in ameloblastomas.

  3. Behavioral stress may increase the rewarding valence of cocaine-associated cues through a dynorphin/kappa-opioid receptor-mediated mechanism without affecting associative learning or memory retrieval mechanisms.

    Science.gov (United States)

    Schindler, Abigail G; Li, Shuang; Chavkin, Charles

    2010-08-01

    Stress exposure increases the risk of addictive drug use in human and animal models of drug addiction by mechanisms that are not completely understood. Mice subjected to repeated forced swim stress (FSS) before cocaine develop significantly greater conditioned place preference (CPP) for the drug-paired chamber than unstressed mice. Analysis of the dose dependency showed that FSS increased both the maximal CPP response and sensitivity to cocaine. To determine whether FSS potentiated CPP by enhancing associative learning mechanisms, mice were conditioned with cocaine in the absence of stress, then challenged after association was complete with the kappa-opioid receptor (KOR) agonist U50,488 or repeated FSS, before preference testing. Mice challenged with U50,488 60 min before CPP preference testing expressed significantly greater cocaine-CPP than saline-challenged mice. Potentiation by U50,488 was dose and time dependent and blocked by the KOR antagonist norbinaltorphimine (norBNI). Similarly, mice subjected to repeated FSS before the final preference test expressed significantly greater cocaine-CPP than unstressed controls, and FSS-induced potentiation was blocked by norBNI. Novel object recognition (NOR) performance was not affected by U50,488 given 60 min before assay, but was impaired when given 15 min before NOR assay, suggesting that KOR activation did not potentiate CPP by facilitating memory retrieval or expression. The results from this study show that the potentiation of cocaine-CPP by KOR activation does not result from an enhancement of associative learning mechanisms and that stress may instead enhance the rewarding valence of cocaine-associated cues by a dynorphin-dependent mechanism.

  4. $\\kappa$-Deformations and Extended $\\kappa$-Minkowski Spacetimes

    CERN Document Server

    Borowiec, Andrzej

    2014-01-01

    We extend our previous study of Hopf-algebraic $\\kappa$-deformations of all inhomogeneous orthogonal Lie algebras ${\\rm iso}(g)$ as written in a tensorial and unified form. Such deformations are determined by a vector $\\tau$ which for Lorentzian signature can be taken time-, light- or space-like. We focus on some mathematical aspects related to this subject. Firstly, we describe real forms with connection to the metric's signatures and their compatibility with the reality condition for the corresponding $\\kappa$-Minkowski (Hopf) module algebras. Secondly, $h$-adic vs $q$-analog (polynomial) versions of deformed algebras including specialization of the formal deformation parameter $\\kappa$ to some numerical value are considered. In the latter the general covariance is lost and one deals with an orthogonal decomposition. The last topic treated in this paper concerns twisted extensions of $\\kappa$-deformations as well as the description of resulting noncommutative spacetime algebras in terms of solvable Lie alge...

  5. Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK.

    Science.gov (United States)

    Castrillo, A; de Las Heras, B; Hortelano, S; Rodriguez, B; Villar, A; Bosca, L

    2001-05-11

    The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.

  6. Requirement of NF-kappaB/Rel for the development of hair follicles and other epidermal appendices.

    Science.gov (United States)

    Schmidt-Ullrich, R; Aebischer, T; Hülsken, J; Birchmeier, W; Klemm, U; Scheidereit, C

    2001-10-01

    NF-kappaB/Rel transcription factors and IkappaB kinases (IKK) are essential for inflammation and immune responses, but also for bone-morphogenesis, skin proliferation and differentiation. Determining their other functions has previously been impossible, owing to embryonic lethality of NF-kappaB/Rel or IKK-deficient animals. Using a gene targeting approach we have ubiquitously expressed an NF-kappaB super-repressor to investigate NF-kappaB functions in the adult. Mice with suppressed NF-kappaB revealed defective early morphogenesis of hair follicles, exocrine glands and teeth, identical to Eda (tabby) and Edar (downless) mutant mice. These affected epithelial appendices normally display high NF-kappaB activity, suppression of which resulted in increased apoptosis, indicating that NF-kappaB acts as a survival factor downstream of the tumor necrosis factor receptor family member EDAR. Furthermore, NF-kappaB is required for peripheral lymph node formation and macrophage function.

  7. Clinical aspects of phage therapy.

    Science.gov (United States)

    Międzybrodzki, Ryszard; Borysowski, Jan; Weber-Dąbrowska, Beata; Fortuna, Wojciech; Letkiewicz, Sławomir; Szufnarowski, Krzysztof; Pawełczyk, Zdzisław; Rogóż, Paweł; Kłak, Marlena; Wojtasik, Elżbieta; Górski, Andrzej

    2012-01-01

    Phage therapy (PT) is a unique method of treatment of bacterial infections using bacteriophages (phages)-viruses that specifically kill bacteria, including their antibiotic-resistant strains. Over the last decade a marked increase in interest in the therapeutic use of phages has been observed, which has resulted from a substantial rise in the prevalence of antibiotic resistance of bacteria, coupled with an inadequate number of new antibiotics. The first, and so far the only, center of PT in the European Union is the Phage Therapy Unit (PTU) established at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland in 2005. This center continues the rich tradition of PT in Poland, which dates from the early 1920s. The main objective of this chapter is to present a detailed retrospective analysis of the results of PT of 153 patients with a wide range of infections resistant to antibiotic therapy admitted for treatment at the PTU between January 2008 and December 2010. Analysis includes the evaluation of both the efficacy and the safety of PT. In general, data suggest that PT can provide good clinical results in a significant cohort of patients with otherwise untreatable chronic bacterial infections and is essentially well tolerated. In addition, the whole complex procedure employed to obtain and characterize therapeutic phage preparations, as well as ethical aspects of PT, is discussed.

  8. Complete Genome Sequence of Vibrio anguillarum Phage CHOED Successfully Used for Phage Therapy in Aquaculture

    OpenAIRE

    Romero, Jaime; Higuera, Gastón; Gajardo,Felipe; Castillo, Daniel; Middleboe, Mathias; García, Katherine; Ramírez, Carolina; Espejo, Romilio T.

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V. anguillarum phage CHOED.

  9. Insight into screening immunoglobulin gene combinatorial libraries in a phage display vector: a tale of two antibodies.

    Science.gov (United States)

    Kakinuma, A; Portolano, S; Chazenbalk, G; Rapoport, B; McLachlan, S M

    1997-01-01

    Combinatorial libraries of immunoglobulin genes in "phage display" vectors are a powerful tool for obtaining antigen-specific antibody fragments. To date, this approach has been used to isolate abundant, but not rare, human autoantibodies of IgG class. We have compared the relative efficiencies of panning pComb3 libraries made from intrathyroidal plasma cells for abundant human autoantibodies to thyroid peroxidase (TPO) and rare autoantibodies to the thyrotropin receptor (TSHR). TPO-specific Fab were readily obtained from a library using three different forms of recombinant antigen, (i) purified TPO, (ii) impure TPO in culture medium and, (iii) TPO expressed on the surface of CHO cells. In contrast, TSHR-specific Fab were not isolated. This was the case despite repeated pannings of six libraries from three optimal patients (IgG/kappa and IgG/lambda libraries for each patient). Both purified recombinant TSHR and CHO cells expressing TSHR on their surface were used. Library enrichment was observed on some screenings. However, Fab expressed by individual clones or from enriched libraries were not specific as determined by (i) binding to purified, radio-labeled antigen, (ii) FACS analysis of TSHR on intact CHO cells and, (iii) inhibition of radiolabeled TSH binding. Remarkably, in screening for both TPO- and TSHR-specific Fab, neither library enrichment nor the retention of cDNA inserts of the correct size correlated with obtaining Fab with the antigenic specificity sought. Indeed, excellent enrichment could be observed with conditioned medium from untransfected cells. Our data suggest that the key to isolating rare antibodies from phage display libraries is not the creation of vast libraries of greater diversity or even the development of more stable vectors. Rather, success in this endeavor appears to require reducing the "noise" of non-specific clones in a moderately sized library.

  10. Information Phage Therapy Research Should Report

    National Research Council Canada - National Science Library

    Stephen T Abedon

    2017-01-01

    .... Clinically these applications are described as phage or bacteriophage therapy. Alternatively, and especially in the treatment of environments, this practice instead may be described as a phage-mediated biocontrol of bacteria...

  11. Antagonists of the kappa opioid receptor.

    Science.gov (United States)

    Urbano, Mariangela; Guerrero, Miguel; Rosen, Hugh; Roberts, Edward

    2014-05-01

    The research community has increasingly focused on the development of OPRK antagonists as pharmacotherapies for the treatment of depression, anxiety, addictive disorders and other psychiatric conditions produced or exacerbated by stress. Short-acting OPRK antagonists have been recently developed as a potential improvement over long-acting prototypic ligands including nor-BNI and JDTic. Remarkably the short-acting LY2456302 is undergoing phase II clinical trials for the augmentation of the antidepressant therapy in treatment-resistant depression. This Letter reviews relevant chemical and pharmacological advances in the identification and development of OPRK antagonists.

  12. Temperature significantly affects the plaquing and adsorption efficiencies of Listeria phages

    Directory of Open Access Journals (Sweden)

    Jeffrey I. Tokman

    2016-05-01

    Full Text Available Listeria-infecting phages are currently being used to control and detect the important foodborne pathogen Listeria monocytogenes; however, the influence of environmental conditions on the interactions between L. monocytogenes and its phages has not been explored in depth. Here, we examined the infective potential of four Listeria phages (two each from the P70-like and P100-like phages of Listeria against five strains of L. monocytogenes (representing serotypes 1/2a, 1/2b, 4a, and 4b grown under a range of temperatures (7ºC – 37°C. We show that the plaquing efficiencies for all four phages were significantly affected by temperature. Interestingly, no plaques were observed for any of the four phages at 37°C. Adsorption assays performed with the P100-like phages, LP-048 and LP-125, showed that LP-048 had a severely reduced adsorption efficiency against susceptible strains at 37°C as compared to 30°C, suggesting that there is considerably less accessible rhamnose (LP-048’s putative phage receptor on the host at 37°C than at 30°C. LP-125 adsorbed to host cells at 37ºC, indicating that the inability for LP-125 to plaque at 37°C is not due to adsorption inhibition. LP-048 showed significantly higher adsorption efficiency against a mutant strain lacking N-acetylglucosamine in its wall teichoic acids than the parental strain at both 30°C and 37°C, suggesting that N-acetylglucosamine competes with rhamnose for glycosylation sites on the wall teichoic acids. The data presented here clearly shows that L. monocytogenes can gain physiological refuge from phage infection, which should be carefully considered for both the design and implementation of phage-based control and detection applications.

  13. Phage Therapy: Eco-Physiological Pharmacology

    OpenAIRE

    Abedon, Stephen T.

    2014-01-01

    Bacterial virus use as antibacterial agents, in the guise of what is commonly known as phage therapy, is an inherently physiological, ecological, and also pharmacological process. Physiologically we can consider metabolic properties of phage infections of bacteria and variation in those properties as a function of preexisting bacterial states. In addition, there are patient responses to pathogenesis, patient responses to phage infections of pathogens, and also patient responses to phage virio...

  14. Phage therapy--constraints and possibilities.

    Science.gov (United States)

    Nilsson, Anders S

    2014-05-01

    The rise of antibiotic-resistant bacterial strains, causing intractable infections, has resulted in an increased interest in phage therapy. Phage therapy preceded antibiotic treatment against bacterial infections and involves the use of bacteriophages, bacterial viruses, to fight bacteria. Virulent phages are abundant and have proven to be very effective in vitro, where they in most cases lyse any bacteria within the hour. Clinical trials on animals and humans show promising results but also that the treatments are not completely effective. This is partly due to the studies being carried out with few phages, and with limited experimental groups, but also the fact that phage therapy has limitations in vivo. Phages are large compared with small antibiotic molecules, and each phage can only infect one or a few bacterial strains. A very large number of different phages are needed to treat infections as these are caused by genetically different strains of bacteria. Phages are effective only if enough of them can reach the bacteria and increase in number in situ. Taken together, this entails high demands on resources for the construction of phage libraries and the testing of individual phages. The effectiveness and host range must be characterized, and immunological risks must be assessed for every single phage.

  15. Synthetic Phage for Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    So Young Yoo

    2014-01-01

    Full Text Available Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

  16. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    Directory of Open Access Journals (Sweden)

    Zhang AL

    2017-02-01

    Full Text Available Ailiang Zhang, Kun Wang, Lianghua Ding, Xinnan Bao, Xuan Wang, Xubin Qiu, Jinbo Liu Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, People’s Republic of China Abstract: Lumbar disc herniation (LDH is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB and nucleotide-binding domain-like receptor protein 3 (NLRP3 inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP was implanted in the left L5 dorsal root ganglion (DRG to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC, caspase-1, interleukin (IL-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. Keywords: pain, NLRP3, NF-κB, dorsal root ganglion, nucleus pulposus

  17. Structure and molecular assignment of lactococcal phage TP901-1 baseplate

    DEFF Research Database (Denmark)

    Bebeacua, Cecilia; Bron, Patrick; Lai, Livia

    2010-01-01

    P335 lactococcal phages infect the Gram(+) bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized...

  18. Phage typing of Staphylococcus saprophyticus.

    Science.gov (United States)

    Torres Pereira, A.; Melo Cristino, J. A.

    1991-01-01

    This study included 502 staphylococcus strains; Staphylococcus saprophyticus (297 strains) S. cohnii (47), S. xylosus (10), S. epidermidis (67) and S. aureus (81). Mitomycin C induction was performed on 100 isolates of S. saprophyticus and all induced strains were reacted with each other. Twenty-six strains proved to be lysogenic. Phages were propagated and titrated. With 12 of the phages there were three frequent associations, named lytic groups A, B and C, which included 75% of all typable strains. Typability of the system was 45% and reproducibility was between 94.2% and 100%. Phages did not lyse S. aureus and S. epidermidis strains, but they lysed S. saprophyticus and only rare strains of other novobiocin resistant species. Effective S. saprophyticus typing serves ecological purposes and tracing the origin of urinary strains from the skin or mucous membranes. Phage typing in association with plasmid profiling previously described, are anticipated as complementary methods with strong discriminatory power for differentiating among S. saprophyticus strains. PMID:1752305

  19. Phage lytic enzymes: a history

    Institute of Scientific and Technical Information of China (English)

    David; Trudil

    2015-01-01

    There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

  20. Phage-Host Interactions in Flavobacterium psychrophilum and the Potential for Phage Therapy in Aquaculture

    DEFF Research Database (Denmark)

    Christiansen, Rói Hammershaimb

    , the increasing problem with antibiotic resistance has led to increased attention to the use of phages for controlling F. psychrophilum infections in aquaculture. In a synopsis and four scientific papers, this PhD project studies the potential and optimizes the use of phage therapy for treatment and prevention...... of F. psychrophilum infections in rainbow trout fry. In the first paper, studies of the controlling effect of different phages infecting F. psychrophilum in liquid cultures showed that a high initial phage concentration was crucial for fast and effective bacterial lysis in the cultures and sensitive...... cells could be maintained at a low level throughout the rest of the experiment. Surprisingly, no difference was observed between infection with single phages or phage cocktails. At the end of incubation phage-sensitive strains dominated in the cultures with low initial phage concentrations and phage...

  1. Injury-induced platelet-derived growth factor receptor-alpha expression mediated by interleukin-1beta (IL-1beta) release and cooperative transactivation by NF-kappaB and ATF-4: IL-1beta facilitates HDAC-1/2 dissociation from promoter.

    Science.gov (United States)

    Zhang, Ning; Khachigian, Levon M

    2009-10-09

    Platelet-derived growth factors are a family of potent mitogens and chemoattractants for fibroblasts and other cells of mesenchymal origin. Platelet-derived growth factor (PDGF) dimeric ligands (composed of A-, B-, C-, and D-chains) exert their biological activity through high affinity interactions with cell surface receptor subunits (alpha and beta). PDGF-receptor-alpha is widely implicated in the pathogenesis of hyperplastic fibrotic disease, yet the molecular mechanisms controlling its expression in response to injury are poorly understood. Here we show that PDGF-R alpha expression is induced in fibroblasts by mechanical injury and interleukin (IL)-1beta, which was abolished by neutralizing IL-1beta antibodies in the culture supernatant or inhibitors of NF-kappaB. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed the existence of a new NF-kappaB binding site at -531/-521 bp in the PDGF-R alpha promoter. We have recently shown that ATF-4 is also induced by injury (Malabanan, K. P., Kanellakis, P., Bobik, A., and Khachigian, L. M. (2008) Circ. Res. 103, 378-387), and we demonstrate here that ATF-4 binds a novel element -259/-254 and stimulates PDGF-R alpha transcription. ATF-4 and NF-kappaB interact, occupy the PDGF-R alpha promoter, and induce PDGF-R alpha transcription in a cooperative manner. IL-1beta facilitates the dissociation of histone deacetylase (HDAC)-1/2 from the PDGF-R alpha promoter, whereas the HDAC inhibitors suberoylanilide hydroxamic acid and trichostatin A potentiate IL-1beta induction of PDGF-R alpha transcription. These findings, taken together, demonstrate that injury stimulates IL-1beta secretion by fibroblasts, which activates NF-kappaB and ATF-4 and stimulates interaction with the PDGF-R alpha promoter, triggering PDGF-R alpha transcription. Physical and functional interactions between NF-kappaB and ATF-4 have not been reported in any gene. This is also the first report of HDAC regulation of PDGF-R alpha

  2. Engineering human interferon α1c/86D with phage display technology

    Institute of Scientific and Technical Information of China (English)

    马学军; 胡荣; 吕海; 魏开坤; 张丽兰; 薛水星; 侯云德

    1999-01-01

    Human interferon-α1c/86D (IFNα1c/86D) was functionally displayed on the surface of the filamentous bacteriophage using a phagemid vector system (pCANTAB5E). The key amino acid residues involved in the receptor binding were further defined with phage displayed 6-mer peptide library and two neutralizing antibodies against linear epitopes on the IFN-α1b, indicating that residues 30, 33, 34, (AB-loop) and residues 124, 126, 127 (D helix, DE-loop) were more critical than the adjacent residues for recognition of receptor. In addition, a cassette mutagenesis library was generated by fully randomizing the sequence of the four positions 29, 31, 32 and 35 in AB-loop, and used to select phage-IFN variants with WISH-hased panning method. Three phage-IFN variants were isolated to possess more antiviral activity in the range of 4—16-fold than parental phage-IFN after IPTG-induced soluble expression. The results suggest that phage displayed phage-IFN α1c/86D variants with increased specific activity might be obta

  3. The habits of highly effective phages: population dynamics as a framework for identifying therapeutic phages

    Directory of Open Access Journals (Sweden)

    James J Bull

    2014-11-01

    Full Text Available The use of bacteriophages as antibacterial agents is being actively researched on a global scale. Typically, the phages used are isolated from the wild by plating on the bacteria of interest, and a far larger set of candidate phages is often available than can be used in any application. When an excess of phages is available, how should the best phages be identified? Here we consider phage-bacterial population dynamics as a basis for evaluating and predicting phage success. A central question is whether the innate dynamical properties of phages are the determinants of success, or instead, whether extrinsic, indirect effects can be responsible. We address the dynamical perspective, motivated in part by the absence of dynamics in previously suggested principles of phage therapy. Current mathematical models of bacterial-phage dynamics do not capture the realities of in vivo dynamics, nor is this likely to change, but they do give insight to qualitative properties that may be generalizable. In particular, phage adsorption rate may be critical to treatment success, so understanding the effects of the in vivo environment on host availability may allow prediction of useful phages prior to in vivo experimentation. Principles for predicting efficacy may be derived by developing a greater understanding of the in vivo system, or such principles could be determined empirically by comparing phages with known differences in their dynamic properties. The comparative approach promises to be a powerful method of discovering the key to phage success. We offer five recommendations for future study: (i compare phages differing in treatment efficacy to identify the phage properties associated with success, (ii assay dynamics in vivo, (iii understand mechanisms of bacterial escape from phages, (iv test phages in model infections that are relevant to the intended clinical applications, and (v develop new classes of models for phage growth in spatially heterogeneous

  4. Phage Display Technology in Biomaterials Engineering: Progress and Opportunities for Applications in Regenerative Medicine.

    Science.gov (United States)

    Martins, Ivone M; Reis, Rui L; Azevedo, Helena S

    2016-11-18

    The field of regenerative medicine has been gaining momentum steadily over the past few years. The emphasis in regenerative medicine is to use various in vitro and in vivo approaches that leverage the intrinsic healing mechanisms of the body to treat patients with disabling injuries and chronic diseases such as diabetes, osteoarthritis, and degenerative disorders of the cardiovascular and central nervous system. Phage display has been successfully employed to identify peptide ligands for a wide variety of targets, ranging from relatively small molecules (enzymes, cell receptors) to inorganic, organic, and biological (tissues) materials. Over the past two decades, phage display technology has advanced tremendously and has become a powerful tool in the most varied fields of research, including biotechnology, materials science, cell biology, pharmacology, and diagnostics. The growing interest in and success of phage display libraries is largely due to its incredible versatility and practical use. This review discusses the potential of phage display technology in biomaterials engineering for applications in regenerative medicine.

  5. The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties.

    Directory of Open Access Journals (Sweden)

    Anneleen Cornelissen

    Full Text Available Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4 and 10(6 pfu and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.

  6. Recombinant phage probes for Listeria monocytogenes

    Energy Technology Data Exchange (ETDEWEB)

    Carnazza, S; Gioffre, G; Felici, F; Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

    2007-10-03

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 10{sup 4} cells ml{sup -1}. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  7. Recombinant phage probes for Listeria monocytogenes

    Science.gov (United States)

    Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

    2007-10-01

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  8. Stress stimulation and signal pathways of osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand in bone metabolism%应力刺激与骨保护蛋白、核因子κB受体活化因子及其配体骨代谢信号通路

    Institute of Scientific and Technical Information of China (English)

    王丹; 鲍捷; 王国祥

    2012-01-01

    背景:骨保护蛋白、核因子κB 受体活化因子及其配体(osteoprote-gerin/ ligand of receptor activator of NF-κB/receptoractivator of NF-κB,OPG/RANKL/RANK)骨代谢信号通路是对应力敏感的通路之一.不同性质的运动会产生不同的机械应力刺激,影响骨代谢信号通路.目的:观察不同性质的运动对OPG/RANKL/RANK 信号通路的影响.方法:由第一作者于2000/2011 通过计算机检索 CNKI,HighWire 和Elsevier 数据库中关于" 应力刺激与OPG/RANKL/RANK"的相关的论文报告.以"应力刺激,OPG/RANKL/RANK"或"应力刺激,骨代谢"为检索词进行检索.选择的文章内容与应力刺激对信号通路的影响有关,选择相关近期发表的文献或者是发表在权威期刊的文献.共检索到215 篇文章,按纳入和排除标准对文献进行筛选,共纳入31 篇文章.结果与结论:运动对骨骼不断产生机械应力刺激,这种机械应力刺激可以通过影响成骨细胞和破骨细胞的OPG/RNAKL/RANK 信号调节系统而调节骨组织代谢.但是相关文献中的研究结果不一致,有待进一步的研究.%BACKGROUND: Signal pathway of osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand in bone metabolismis one of the accesses to cope with the stress sensitivity. Different types of the exercise can produce different types of stressstimulation, and influence the signal pathways in bone metabolism.OBJECTIVE: To explore the influence of different types of exercise on the signal pathways of osteoprotegerin, receptor activator ofnuclear factor kappa B and its ligand in bone metabolism.METHODS: An online search of CNKI, HighWire and Elsevier databases was performed for articles related to "stress stimulation,osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand". Articles published from 2000 to 2011 with thekeywords of "stress stimulation, osteoprotegerin, receptor activator of nuclear factor kappa B and its ligand" or

  9. Trackable and Targeted Phage as Positron Emission Tomography (PET Agent for Cancer Imaging

    Directory of Open Access Journals (Sweden)

    Zibo Li, Qiaoling Jin, Chiunwei Huang, Siva Dasa, Liaohai Chen, Li-peng Yap, Shuanglong Liu, Hancheng Cai, Ryan Park, Peter S Conti

    2011-01-01

    Full Text Available The recent advancement of nanotechnology has provided unprecedented opportunities for the development of nanoparticle enabled technologies for detecting and treating cancer. Here, we reported the construction of a PET trackable organic nanoplatform based on phage particle for targeted tumor imaging. Method: The integrin αvβ3 targeted phage nanoparticle was constructed by expressing RGD peptides on its surface. The target binding affinity of this engineered phage particle was evaluated in vitro. A bifunctional chelator (BFC 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA or 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino methyl benzoic acid (AmBaSar was then conjugated to the phage surface for 64Cu2+ chelation. After 64Cu radiolabeling, microPET imaging was performed in U87MG tumor model and the receptor specificity was confirmed by blocking experiments. Results: The phage-RGD demonstrated target specificity based on ELISA experiment. According to the TEM images, the morphology of the phage was unchanged after the modification with BFCs. The labeling yield was 25 ± 4% for 64Cu-DOTA-phage-RGD and 46 ± 5% for 64Cu-AmBaSar-phage-RGD, respectively. At 1 h time point, 64Cu-DOTA-phage-RGD and 64Cu-AmBaSar-phage-RGD have comparable tumor uptake (~ 8%ID/g. However, 64Cu-AmBaSar-phage-RGD showed significantly higher tumor uptake (13.2 ± 1.5 %ID/g, P<0.05 at late time points compared with 64Cu-DOTA-phage-RGD (10 ± 1.2 %ID/g. 64Cu-AmBaSar-phage-RGD also demonstrated significantly lower liver uptake, which could be attributed to the stability difference between these chelators. There is no significant difference between two tracers regarding the uptake in kidney and muscle at all time points tested. In order to confirm the receptor specificity, blocking experiment was performed. In the RGD blocking experiment, the cold RGD peptide was injected 2 min before the administration of 64Cu-AmBaSar-phage-RGD. Tumor uptake was

  10. Last of the T Phages

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F. W.

    1978-01-01

    Results clearly show that it is possible to induce mutations in T7 DNA at a physically measurable rate in the laboratory, and to follow genetic divergence by restriction analysis. The rate of accumulation of changes in the presence of mutagen is high enough that it may be feasible to induce changes at least as great as those found among the T7-related phages isolated from nature.

  11. Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection.

    Science.gov (United States)

    Yao, Virginia J; Ozawa, Michael G; Trepel, Martin; Arap, Wadih; McDonald, Donald M; Pasqualini, Renata

    2005-02-01

    Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.

  12. High affinity antibodies against Lex and sialyl Lex from a phage display library.

    Science.gov (United States)

    Dinh, Q; Weng, N P; Kiso, M; Ishida, H; Hasegawa, A; Marcus, D M

    1996-07-15

    Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.

  13. An Automation Interface for Kappa PC

    DEFF Research Database (Denmark)

    Hartvig, Susanne C

    1999-01-01

    The reports documents an automation interface for Kappa PC. The automation interface can be used to embed Kappa applications in 32-bit Windowsapplications.The interface includes functions for initialising Kappa, for loading an application, for settingvalues, for getting values, and for stopping...

  14. An Automation Interface for Kappa PC

    DEFF Research Database (Denmark)

    Hartvig, Susanne C

    1999-01-01

    The reports documents an automation interface for Kappa PC. The automation interface can be used to embed Kappa applications in 32-bit Windowsapplications.The interface includes functions for initialising Kappa, for loading an application, for settingvalues, for getting values, and for stopping...

  15. The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808

    Directory of Open Access Journals (Sweden)

    Patricia eMunsch-Alatossava

    2013-12-01

    Full Text Available The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lb. delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimise the risks associated with the appearance and attack of phages in the manufacture of yoghurt, and Swiss or Italian type hard cheeses, which typically use thermophilic LAB starter cultures containing Lb. delbrueckii strains among others. This mini review article summarises the present data concerning (i the special features, particle structure and components of phage LL-H and (ii the structure and properties of lipoteichoic acids (LTAs, which are the phage LL-H receptor components of Lb. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of Lb. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

  16. Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails.

    Science.gov (United States)

    Pereira, Carla; Moreirinha, Catarina; Lewicka, Magdalena; Almeida, Paulo; Clemente, Carla; Cunha, Ângela; Delgadillo, Ivonne; Romalde, Jésus L; Nunes, Maria L; Almeida, Adelaide

    2016-07-15

    The aim of this study was to compare the dynamics of three previously isolated bacteriophages (or phages) individually (phSE-1, phSE-2 and phSE-5) or combined in cocktails of two or three phages (phSE-1/phSE-2, phSE-1/phSE-5, phSE-2/phSE-5 and phSE-1/phSE-2/phSE-5) to control Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in order to evaluate their potential application during depuration. Phages were assigned to the family Siphoviridae and revealed identical restriction digest profiles, although they showed a different phage adsorption, host range, burst size, explosion time and survival in seawater. The three phages were effective against S. Typhimurium (reduction of ∼2.0 log CFU/mL after 4h treatment). The use of cocktails was not significantly more effective than the use of single phages. A big fraction of the remained bacteria are phage-resistant mutants (frequency of phage-resistant mutants 9.19×10(-5)-5.11×10(-4)) but phage- resistant bacterial mutants was lower for the cocktail phages than for the single phage suspensions and the phage phSE-1 presented the highest rate of resistance and phage phSE-5 the lowest one. The spectral changes of S. Typhimurium resistant and phage-sensitive cells were compared and revealed relevant differences for peaks associated to amide I (1620cm(-1)) and amide II (1515cm(-1)) from proteins and from carbohydrates and phosphates region (1080-1000cm(-1)). Despite the similar efficiency of individual phages, the development of lower resistance indicates that phage cocktails might be the most promising choice to be used during the bivalve depuration to control the transmission of salmonellosis.

  17. Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy

    OpenAIRE

    Maciej Żaczek; Marzanna Łusiak-Szelachowska; Ewa Jończyk-Matysiak; Beata Weber-Dąbrowska; Ryszard Międzybrodzki; Barbara Owczarek; Agnieszka Kopciuch; Wojciech Fortuna; Paweł Rogóż; Andrzej Górski

    2016-01-01

    In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties towards applied phages (K rate). Among 20 examined patients receiving...

  18. Phage type and sensitivity to antibiotics of Staphylococcus aureus film-forming strains isolated from airway mucosa

    Directory of Open Access Journals (Sweden)

    O. S. Voronkova

    2014-10-01

    indicate possible differences in the structure of the cell wall, composition of the cytoplasmic membrane, receptors, etc. So, phage typing may be used as one of tests to study film-forming processes. Besides, phage typing can be helpful in finding of the source and modes of transmission of nosocomial strains of bacteria. Given the increased level of antibiotic resistance among film-forming strains of staphylococci, the importance of this monitoring is indisputable, because spreading of these strains may cause the failure of therapy.

  19. Use of phages to control Campylobacter spp.

    Science.gov (United States)

    Janež, Nika; Loc-Carrillo, Catherine

    2013-10-01

    The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo.

  20. [Peptide phage display in biotechnology and biomedicine].

    Science.gov (United States)

    Kuzmicheva, G A; Belyavskaya, V A

    2016-07-01

    To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

  1. Information Phage Therapy Research Should Report.

    Science.gov (United States)

    Abedon, Stephen T

    2017-04-30

    Bacteriophages, or phages, are viruses which infect bacteria. A large subset of phages infect bactericidally and, consequently, for nearly one hundred years have been employed as antibacterial agents both within and outside of medicine. Clinically these applications are described as phage or bacteriophage therapy. Alternatively, and especially in the treatment of environments, this practice instead may be described as a phage-mediated biocontrol of bacteria. Though the history of phage therapy has involved substantial clinical experimentation, current standards along with drug regulations have placed a premium on preclinical approaches, i.e., animal experiments. As such, it is important for preclinical experiments not only to be held to high standards but also to be reported in a manner which improves translation to clinical utility. Here I address this latter issue, that of optimization of reporting of preclinical as well as clinical experiments. I do this by providing a list of pertinent information and data which, in my opinion, phage therapy experiments ought to present in publications, along with tips for best practices. The goal is to improve the ability of readers to gain relevant information from reports on phage therapy research, to allow other researchers greater potential to repeat or extend findings, to ease transitions from preclinical to clinical development, and otherwise simply to improve phage therapy experiments. Targeted are not just authors but also reviewers, other critical readers, writers of commentaries, and, perhaps, formulators of guidelines or policy. Though emphasizing therapy, many points are applicable to phage-mediated biocontrol of bacteria more generally.

  2. DMPD: Nuclear factor-kappaB: activation and regulation during toll-like receptorsignaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17349209 Nuclear factor-kappaB: activation and regulation during toll-like receptor...signaling. Carmody RJ, Chen YH. Cell Mol Immunol. 2007 Feb;4(1):31-41. (.png) (.svg) (.html) (.csml) Show Nuclear fact...or-kappaB: activation and regulation during toll-like receptorsignaling. PubmedID 17349209 Title Nuclear fact

  3. 金雀异黄素对类风湿关节炎患者成纤维样滑膜细胞骨保护素/细胞核因子κB受体活化因子/细胞核因子κB受体活化因子配体通路的影响%Effect of Genistein on the Osteoprotegerin/Receptor-activator of Nuclear Factor Kappa Bata/Receptor-activator of Nuclear Factor Kappa Bata Ligand System of Fibroblast-like Synoviocytes

    Institute of Scientific and Technical Information of China (English)

    王燕茹; 张育; 沈维干; 尚辰; 马莹莹; 方振玉

    2013-01-01

    目的 研究金雀异黄素(genistein,Gen)对人类风湿关节炎(rheumatoid arthritis,RA)成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)骨保护素(osteoprotegerin,OPG)/细胞核因子κB受体活化因子(receptor-activator of nuclear factor kappa bata,RANK)/细胞核因子κB受体活化因子配体(receptor-activator of nuclear factor kappa bata ligand,RANKL)通路的影响及机制.方法 培养人RA-FLS,并分为对照组(RA-FLS细胞)、TNF-α组(RA-FLS细胞+TNF-α 10 ng/ml)、TNF-α+ Gen组(RA-FLS细胞+TNF-α 10 ng/ml+ Gen 50 μM)、Gen组(RA-FLS细胞+Gen 50 μM).应用免疫印迹检测Gen作用RA-FLS的RANK、RANKL、OPG、雌激素受体(estrogen receptor α,ERα)表达情况,免疫荧光检测ERα细胞内分布情况.结果 Gen组、TNF-α组及TNF-α+ Gen组细胞内RANK蛋白表达量较对照组无明显差异(P>0.05),但OPG及RANKL蛋白表达明显高于对照组(均P<0.05).Gen组OPG/RANKL较对照组增高(P<0.05).Gen组、TNF-α组及TNF-α+ Gen组细胞内ERα蛋白表达量与对照组比较,差异无统计学意义(P>0.05).免疫荧光结果显示核内ERα表达量增加.结论 Gen可能通过与雌激素受体结合,转入核内发挥免疫调节作用,从而调节OPG/RANKL/RANK通路,发挥抑制骨破坏的作用.Gen可以上调人RA-FLS中OPG及RANKL,且对OPG的上调作用更强.

  4. Selection of phages and conditions for the safe phage therapy against Pseudomonas aeruginosa infections.

    Science.gov (United States)

    Krylov, Victor; Shaburova, Olga; Pleteneva, Elena; Krylov, Sergey; Kaplan, Alla; Burkaltseva, Maria; Polygach, Olga; Chesnokova, Elena

    2015-02-01

    The emergence of multidrug-resistant bacterial pathogens forced us to consider the phage therapy as one of the possible alternative approaches to treatment. The purpose of this paper is to consider the conditions for the safe, long-term use of phage therapy against various infections caused by Pseudomonas aeruginosa. We describe the selection of the most suitable phages, their most effective combinations and some approaches for the rapid recognition of phages unsuitable for use in therapy. The benefits and disadvantages of the various different approaches to the preparation of phage mixtures are considered, together with the specific conditions that are required for the safe application of phage therapy in general hospitals and the possibilities for the development of personalized phage therapy.

  5. Phage-Phagocyte Interactions and Their Implications for Phage Application as Therapeutics

    Directory of Open Access Journals (Sweden)

    Ewa Jończyk-Matysiak

    2017-06-01

    Full Text Available Phagocytes are the main component of innate immunity. They remove pathogens and particles from organisms using their bactericidal tools in the form of both reactive oxygen species and degrading enzymes—contained in granules—that are potentially toxic proteins. Therefore, it is important to investigate the possible interactions between phages and immune cells and avoid any phage side effects on them. Recent progress in knowledge concerning the influence of phages on phagocytes is also important as such interactions may shape the immune response. In this review we have summarized the current knowledge on phage interactions with phagocytes described so far and their potential implications for phage therapy. The data suggesting that phage do not downregulate important phagocyte functions are especially relevant for the concept of phage therapy.

  6. Selection of phages and conditions for the safe phage therapy against Pseudomonas aeruginosa infections

    Institute of Scientific and Technical Information of China (English)

    Victor; Krylov; Olga; Shaburova; Elena; Pleteneva; Sergey; Krylov; Alla; Kaplan; Maria; Burkaltseva; Olga; Polygach; Elena; Chesnokova

    2015-01-01

    The emergence of multidrug-resistant bacterial pathogens forced us to consider the phage therapy as one of the possible alternative approaches to treatment. The purpose of this paper is to consider the conditions for the safe, long-term use of phage therapy against various infections caused by Pseudomonas aeruginosa. We describe the selection of the most suitable phages, their most effective combinations and some approaches for the rapid recognition of phages unsuitable for use in therapy. The benefi ts and disadvantages of the various different approaches to the preparation of phage mixtures are considered, together with the specifi c conditions that are required for the safe application of phage therapy in general hospitals and the possibilities for the development of personalized phage therapy.

  7. A shortcut in phage screening technique

    Directory of Open Access Journals (Sweden)

    Alexandre de Andrade

    2005-03-01

    Full Text Available A simple modification of the traditional Benton & Davis technique for phage screening is presented that avoids the tedious sample dilutions of putative spots/phages towards the second screening. With the use of a sole agar plate and nylon filter, the modification distinguishes a true positive recombinant from a false positive, with high probability of success.

  8. Methods for Selecting Phage Display Antibody Libraries.

    Science.gov (United States)

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  9. Aeromonas phages encode tRNAs for their overused codons.

    Science.gov (United States)

    Prabhakaran, Ramanandan; Chithambaram, Shivapriya; Xia, Xuhua

    2014-01-01

    The GC-rich bacterial species, Aeromonas salmonicida, is parasitised by both GC-rich phages (Aeromonas phages - phiAS7 and vB_AsaM-56) and GC-poor phages (Aeromonas phages - 25, 31, 44RR2.8t, 65, Aes508, phiAS4 and phiAS5). Both the GC-rich Aeromonas phage phiAS7 and Aeromonas phage vB_AsaM-56 have nearly identical codon usage bias as their host. While all the remaining seven GC-poor Aeromonas phages differ dramatically in codon usage from their GC-rich host. Here, we investigated whether tRNA encoded in the genome of Aeromonas phages facilitate the translation of phage proteins. We found that tRNAs encoded in the phage genome correspond to synonymous codons overused in the phage genes but not in the host genes.

  10. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib......CP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain...... reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody...

  11. Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

    DEFF Research Database (Denmark)

    Hougs, L; Barington, T; Madsen, HO

    1993-01-01

    reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody......CP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain...... of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the Hib...

  12. Structure, adsorption to host, and infection mechanism of virulent lactococcal phage p2.

    Science.gov (United States)

    Bebeacua, Cecilia; Tremblay, Denise; Farenc, Carine; Chapot-Chartier, Marie-Pierre; Sadovskaya, Irina; van Heel, Marin; Veesler, David; Moineau, Sylvain; Cambillau, Christian

    2013-11-01

    Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower k(off) values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.

  13. Hawking radiation in the kappa-spacetime

    CERN Document Server

    Harikumar, E

    2016-01-01

    In this paper, we analyze the Hawking radiation of kappa-deformed Schwarzchild black hole and obtain the deformed Hawking temperature. For this, we first derive deformed metric for the kappa-spacetime, which in the generic case, is not a symmetric tensor and also has a momentum dependence. We show that the Schwarzchild metric obtained in the kappa-deformed spacetime has a dependence on energy. We use the fact that the deformed metric is conformally flat in the 1+1 dimensions, to solve the kappa-deformed Klein-Gordon equation in the background of the Schwarzchild metric. The method of Boguliobov coefficients is then used to calculate the thermal spectrum of kappa-deformed-Schwarzchild black hole and show that the Hawking temperature is modified by the non-commutativity of the kappa-spacetime.

  14. Information Phage Therapy Research Should Report

    Directory of Open Access Journals (Sweden)

    Stephen T. Abedon

    2017-04-01

    Full Text Available Bacteriophages, or phages, are viruses which infect bacteria. A large subset of phages infect bactericidally and, consequently, for nearly one hundred years have been employed as antibacterial agents both within and outside of medicine. Clinically these applications are described as phage or bacteriophage therapy. Alternatively, and especially in the treatment of environments, this practice instead may be described as a phage-mediated biocontrol of bacteria. Though the history of phage therapy has involved substantial clinical experimentation, current standards along with drug regulations have placed a premium on preclinical approaches, i.e., animal experiments. As such, it is important for preclinical experiments not only to be held to high standards but also to be reported in a manner which improves translation to clinical utility. Here I address this latter issue, that of optimization of reporting of preclinical as well as clinical experiments. I do this by providing a list of pertinent information and data which, in my opinion, phage therapy experiments ought to present in publications, along with tips for best practices. The goal is to improve the ability of readers to gain relevant information from reports on phage therapy research, to allow other researchers greater potential to repeat or extend findings, to ease transitions from preclinical to clinical development, and otherwise simply to improve phage therapy experiments. Targeted are not just authors but also reviewers, other critical readers, writers of commentaries, and, perhaps, formulators of guidelines or policy. Though emphasizing therapy, many points are applicable to phage-mediated biocontrol of bacteria more generally.

  15. Rapid enumeration of phage in monodisperse emulsions.

    Science.gov (United States)

    Tjhung, Katrina F; Burnham, Sean; Anany, Hany; Griffiths, Mansel W; Derda, Ratmir

    2014-06-17

    Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

  16. CARMA1 is required for Akt-mediated NF-kappaB activation in T cells.

    Science.gov (United States)

    Narayan, Preeti; Holt, Brittany; Tosti, Richard; Kane, Lawrence P

    2006-03-01

    Many details of the generic pathway for induction of NF-kappaB have been delineated, but it is still not clear how multiple, diverse receptor systems are able to converge on this evolutionarily conserved family of transcription factors. Recent studies have shown that the CARMA1, Bcl10, and MALT1 proteins are critical for coupling the common elements of the NF-kappaB pathway to the T-cell receptor (TCR) and CD28. We previously demonstrated a role for the serine/threonine kinase Akt in CD28-mediated NF-kappaB induction. Using a CARMA1-deficient T-cell line, we have now found that the CARMA complex is required for induction of NF-kappaB by Akt, in cooperation with protein kinase C activation. Furthermore, using a novel selective inhibitor of Akt, we confirm that Akt plays a modulatory role in NF-kappaB induction by the TCR and CD28. Finally, we provide evidence for a physical and functional interaction between Akt and CARMA and for Akt-dependent phosphorylation of Bcl10. Therefore, in T cells, Akt impinges upon NF-kappaB signaling through at least two separate mechanisms.

  17. High throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface.

    Science.gov (United States)

    Rojas, Gertrudis; Tundidor, Yaima; Infante, Yanelys Cabrera

    2014-01-01

    Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.

  18. Phage-host interaction in Lactococcus and Leuconostoc

    DEFF Research Database (Denmark)

    Kot, Witold Piotr

    Lactic acid bacteria (LAB) are economically important group of microbes used as starter cultures in the dairy industry. Bacteriophages attacking LAB are the main factor responsible for fermentations failures, thus creating significant economical losses worldwide. In the first part of this study......, bacteriophages infecting Leuconostoc species were characterized. A large group of Leuconostoc phages was characterized regarding the morphology, host range variation and genetic relatedness. A classification system for dairy Leuconostoc phages was proposed. dditionally, a PCR test for the dairy Leuconostoc...... corresponds with the species of the host. The two groups shared overall genomic organization but differed significantly on the nucleotide level. One of the specific genes, presumably coding for the receptor binding protein (RBP) in Leuconostoc pseudomesenteroides phages, was functionally characterized...

  19. Mammalian Host-Versus-Phage immune response determines phage fate in vivo

    OpenAIRE

    Katarzyna Hodyra-Stefaniak; Paulina Miernikiewicz; Jarosław Drapała; Marek Drab; Ewa Jończyk-Matysiak; Dorota Lecion; Zuzanna Kaźmierczak; Weronika Beta; Joanna Majewska; Marek Harhala; Barbara Bubak; Anna Kłopot; Andrzej Górski; Krystyna Dąbrowska

    2015-01-01

    Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluor...

  20. Crocodylian nuclear factor kappa B.

    Science.gov (United States)

    Merchant, Mark; Morkotinis, Vasileios; Hale, Amber; White, Mary; Moran, Chris

    2017-11-01

    We deduced the amino acid (aa) sequence of the nuclear factor kappa B (NFκB) protein from genomic data for the American alligator (Alligator mississippiensis), the estuarine crocodile (Crocodylus porosus), and the Indian gharial (Gavialis gangeticus). A 105kDa protein, NFκB1 exhibits complex post-translational processing, multiple mechanisms of activation, and acts as precursor for a p50, a Rel homology transcription factor which influences the expression of key genes for developmental processes, apoptosis, and immune function. The aa sequences of the crocodylian proteins share very high identity with each other (97.2±0.7%), birds (81.0±1.1%, n=6), mammals (75.3±1.6%, n=4), reptiles (80.3±5.1%, n=2), and less identity with fish (55.5±5.5%, n=4) and one amphibian (66.1±0.8%). The crocodylian protein has a well-conserved Rel homology domain, a nuclear localization signal, and a glycine-rich region which facilitates proteasome-mediated generation of p50. The Rel homology domain contains sequences responsible for dimerization, DNA-binding, and nuclear translocation. In addition, seven ankyrin repeats were located, which putatively allow for inhibition of transcriptional regulation by mediating interaction with Inhibitor kappa B. Other features include a death domain, and conserved serine residues, near the C-terminal end, which act as potential phosphorylation sites for activation of the proteolytic generation of p50. Western blot analysis showed both the 105kDa precursor and the 50kDa mature NFκB were expressed in the alligator liver. Nuclear factor κB exhibited diffuse cytoplasmic distribution in alligator hepatocytes, and almost no cytoplasmic localization in infected animals. In addition, nuclear NFκB exhibited specific binding to the consensus NFκB promoter element. Published by Elsevier Inc.

  1. Phage as a modulator of immune responses: practical implications for phage therapy.

    Science.gov (United States)

    Górski, Andrzej; Międzybrodzki, Ryszard; Borysowski, Jan; Dąbrowska, Krystyna; Wierzbicki, Piotr; Ohams, Monika; Korczak-Kowalska, Grażyna; Olszowska-Zaremba, Natasza; Łusiak-Szelachowska, Marzena; Kłak, Marlena; Jończyk, Ewa; Kaniuga, Ewelina; Gołaś, Aneta; Purchla, Sylwia; Weber-Dąbrowska, Beata; Letkiewicz, Sławomir; Fortuna, Wojciech; Szufnarowski, Krzysztof; Pawełczyk, Zdzisław; Rogóż, Paweł; Kłosowska, Danuta

    2012-01-01

    Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

  2. Phage-Antibiotic Synergy (PAS): beta-lactam and quinolone antibiotics stimulate virulent phage growth.

    Science.gov (United States)

    Comeau, André M; Tétart, Françoise; Trojet, Sabrina N; Prère, Marie-Françoise; Krisch, H M

    2007-08-29

    Although the multiplication of bacteriophages (phages) has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cell's production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strain's production of the phage PhiMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS). A related effect was observed in diverse host-phage systems, including the T4-like phages, with beta-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages.

  3. Phage-Antibiotic Synergy (PAS: beta-lactam and quinolone antibiotics stimulate virulent phage growth.

    Directory of Open Access Journals (Sweden)

    André M Comeau

    Full Text Available Although the multiplication of bacteriophages (phages has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cell's production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strain's production of the phage PhiMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS. A related effect was observed in diverse host-phage systems, including the T4-like phages, with beta-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages.

  4. Phage Display Derived IgNAR V Region Binding Domains for Therapeutic Development.

    Science.gov (United States)

    Ubah, Obinna C; Barelle, Caroline J; Buschhaus, Magdalena J; Porter, Andrew J

    2016-01-01

    Phage display technology has revolutionized the science of drug discovery by transforming the generation and manipulation of ligands, such as antibody fragments, enzymes, and peptides. The basis of this technology is the expression of recombinant proteins or peptides fused to a phage coat protein, and subsequent isolation of ligands based on a variety of catalytic, physicochemical/binding kinetic and/or biological characteristics. An incredible number of diagnostic and therapeutic domains have been successfully isolated using phage display technology. The variable domain of the New Antigen Receptors (VNAR) found in cartilaginous fish, is also amenable to phage display selection. Whilst not an antibody, VNARs are unquestionable the oldest (450 million years), and smallest antigen binding, single-domains so far identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established, enhancing our understanding of the evolutionary origins of humoral immunity and the unusual but divergent ancestry of the VNARs themselves. VNARs exhibit remarkable physicochemical properties, such as small size, stability in extreme conditions, solubility, molecular flexibility, high affinity and selectivity for target. The purpose of this review is to illustrate the important role phage display has played in the isolation and characterization of potent therapeutic and diagnostic VNAR domains.

  5. Genome Sequence of Mycobacterium Phage Waterfoul

    Science.gov (United States)

    Jackson, Paige N.; Embry, Ella K.; Johnson, Christa O.; Watson, Tiara L.; Weast, Sayre K.; DeGraw, Caroline J.; Douglas, Jessica R.; Sellers, J. Michael; D’Angelo, William A.

    2016-01-01

    Waterfoul is a newly isolated temperate siphovirus of Mycobacterium smegmatis mc2155. It was identified as a member of the K5 cluster of Mycobacterium phages and has a 61,248-bp genome with 95 predicted genes. PMID:27856585

  6. Supersize me: Cronobacter sakazakii phage GAP32

    Energy Technology Data Exchange (ETDEWEB)

    Abbasifar, Reza; Griffiths, Mansel W. [Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, Canada N1G 2W1 (Canada); Sabour, Parviz M. [Agriculture and Agri-Food Canada, Guelph Food Research Centre, Guelph, ON, Canada N1G 5C9 (Canada); Ackermann, Hans-Wolfgang [Department of Microbiology-Infectiology and Immunology, Faculty of Medicine, Université Laval, Quebec, QC (Canada); Vandersteegen, Katrien; Lavigne, Rob [Laboratory of Gene Technology, Katholieke Universiteit Leuven, Leuven (Belgium); Noben, Jean-Paul [Biomedical Research Institute and Transnational University Limburg, School of Life Sciences, Hasselt University, Diepenbeek (Belgium); Alanis Villa, Argentina; Abbasifar, Arash [Canadian Research Institute for Food Safety, University of Guelph, Guelph, ON, Canada N1G 2W1 (Canada); Nash, John H.E. [Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, Guelph, ON, Canada N1G 3W4 (Canada); Kropinski, Andrew M., E-mail: akropins@uoguelph.ca [Public Health Agency of Canada, Laboratory for Foodborne Zoonoses, Guelph, ON, Canada N1G 3W4 (Canada); Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1 (Canada)

    2014-07-15

    Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB{sub C}saM{sub G}AP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. - Highlights: • Cronobacter sakazakii phage vB{sub C}saM{sub G}AP32 has a genome of 358,663 bp. • It encodes 545 proteins which is more than Mycoplasma genitalium G37. • It is a member of the Myoviridae. • It is peripherally related to coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2. • GAP32 encodes a chromosome condensation protein.

  7. The Staphylococci Phages Family: An Overview

    Directory of Open Access Journals (Sweden)

    Laurence Van Melderen

    2012-11-01

    Full Text Available Due to their crucial role in pathogenesis and virulence, phages of Staphylococcus aureus have been extensively studied. Most of them encode and disseminate potent staphylococcal virulence factors. In addition, their movements contribute to the extraordinary versatility and adaptability of this prominent pathogen by improving genome plasticity. In addition to S. aureus, phages from coagulase-negative Staphylococci (CoNS are gaining increasing interest. Some of these species, such as S. epidermidis, cause nosocomial infections and are therefore problematic for public health. This review provides an overview of the staphylococcal phages family extended to CoNS phages. At the morphological level, all these phages characterized so far belong to the Caudovirales order and are mainly temperate Siphoviridae. At the molecular level, comparative genomics revealed an extensive mosaicism, with genes organized into functional modules that are frequently exchanged between phages. Evolutionary relationships within this family, as well as with other families, have been highlighted. All these aspects are of crucial importance for our understanding of evolution and emergence of pathogens among bacterial species such as Staphylococci.

  8. Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis

    OpenAIRE

    Plaut, Roger D; Beaber, John W.; Zemansky, Jason; Kaur, Ajinder P.; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A; Mokashi, Vishwesh; Hannah, Ryan M.; Pope, Robert K.; Read, Timothy D.; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

    2014-01-01

    In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles assoc...

  9. Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A, spo0B, and spo0F in Phage AP50c Infection of Bacillus anthracis

    OpenAIRE

    Roger D Plaut; Beaber, John W.; Zemansky, Jason; Kaur, Ajinder P.; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A.; Mokashi, Vishwesh; Hannah, Ryan M.; Pope, Robert K.; Timothy D. Read; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

    2014-01-01

    In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles assoc...

  10. Cardiorenal Effects of Kappa Opioid Peptides During Ontogeny

    Directory of Open Access Journals (Sweden)

    Wei Qi

    2011-01-01

    Full Text Available This review focuses on the physiological roles for kappa opioid receptors (KORs in adult animals and humans, as well as in the developing newborn animal. Our recent findings have provided new information that under physiological conditions in conscious newborn animals, activation of KORs with the selective agonist, U-50488H, results in an aquaresis, as previously observed in adult animals and humans. In addition, we have shown in conscious lambs that KORs modulate systemic and renal haemodynamics as well as the arterial baroreflex control of heart rate, providing a previously unidentified role for KORs.

  11. Colonisation of a phage susceptible Campylobacter jejuni population in two phage positive broiler flocks.

    Directory of Open Access Journals (Sweden)

    Sophie Kittler

    Full Text Available The pathogens Campylobacter jejuni and Campylobacter coli are commensals in the poultry intestine and campylobacteriosis is one of the most frequent foodborne diseases in developed and developing countries. Phages were identified to be effective in reducing intestinal Campylobacter load and this was evaluated, in the first field trials which were recently carried out. The aim of this study was to further investigate Campylobacter population dynamics during phage application on a commercial broiler farm. This study determines the superiority in colonisation of a Campylobacter type found in a field trial that was susceptible to phages in in vitro tests. The colonisation factors, i.e. motility and gamma glutamyl transferase activity, were increased in this type. The clustering in phylogenetic comparisons of MALDI-TOF spectra did not match the ST, biochemical phenotype and phage susceptibility. Occurrence of Campylobacter jejuni strains and phage susceptibility types with different colonisation potential seem to play a very important role in the success of phage therapy in commercial broiler houses. Thus, mechanisms of both, phage susceptibility and Campylobacter colonisation should be further investigated and considered when composing phage cocktails.

  12. Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage

    Science.gov (United States)

    Ma, Yanyan; Li, Erna; Qi, Zhizhen; Li, Huan; Wei, Xiao; Lin, Weishi; Zhao, Ruixiang; Jiang, Aimin; Yang, Huiying; Yin, Zhe; Yuan, Jing; Zhao, Xiangna

    2016-01-01

    Achromobacter xylosoxidans, an opportunistic pathogen, is responsible for various nosocomial and community-acquired infections. We isolated phiAxp-3, an N4-like bacteriophage that infects A. xylosoxidans, from hospital waste and studied its genomic and biological properties. Transmission electron microscopy revealed that, with a 67-nm diameter icosahedral head and a 20-nm non-contractile tail, phiAxp-3 has features characteristic of Podoviridae bacteriophages (order Caudovirales). With a burst size of 9000 plaque-forming units and a latent period of 80 min, phiAxp-3 had a host range limited to only four A. xylosoxidans strains of the 35 strains that were tested. The 72,825 bp phiAxp-3 DNA genome, with 416-bp terminal redundant ends, contains 80 predicted open reading frames, none of which are related to virulence or drug resistance. Genome sequence comparisons place phiAxp-3 more closely with JWAlpha and JWDelta Achromobacter phages than with other N4 viruses. Using proteomics, we identified 25 viral proteins from purified phiAxp-3 particles. Notably, investigation of the phage phiAxp-3 receptor on the surface of the host cell revealed that lipopolysaccharide serves as the receptor for the adsorption of phage phiAxp-3. Our findings advance current knowledge about A. xylosoxidans phages in an age where alternative therapies to combat antibiotic-resistant bacteria are urgently needed. PMID:27094846

  13. Involvement of nuclear factor {kappa}B in platelet CD40 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Hachem, Ahmed [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Yacoub, Daniel [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Zaid, Younes [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Mourad, Walid [Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada); Centre Hospitalier Universite de Montreal, 264 boul. Rene-Levesque est, Montreal, Quebec, Canada H2X 1P1 (Canada); Merhi, Yahye, E-mail: yahye.merhi@icm-mhi.org [Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, 5000 Belanger, Montreal, Quebec, Canada H1T 1C8 (Canada); Universite de Montreal, Department of Medicine, 2900 boul. Edouard-Montpetit, Montreal, Quebec, Canada H3T 1J4 (Canada)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer sCD40L induces TRAF2 association to CD40 and NF-{kappa}B activation in platelets. Black-Right-Pointing-Pointer I{kappa}B{alpha} phosphorylation downstream of CD40L/CD40 signaling is independent of p38 MAPK phosphorylation. Black-Right-Pointing-Pointer I{kappa}B{alpha} is required for sCD40L-induced platelet activation and potentiation of aggregation. -- Abstract: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to potentiate platelet activation and aggregation, in a CD40-dependent manner, via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In many cells, the CD40L/CD40 dyad also induces activation of nuclear factor kappa B (NF-{kappa}B). Given that platelets contain NF-{kappa}B, we hypothesized that it may be involved in platelet CD40 signaling and function. In human platelets, sCD40L induces association of CD40 with its adaptor protein the tumor necrosis factor receptor associated factor 2 and triggers phosphorylation of I{kappa}B{alpha}, which are abolished by CD40L blockade. Inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced I{kappa}B{alpha} phosphorylation without affecting p38 MAPK phosphorylation. On the other hand, inhibition of p38 MAPK phosphorylation has no effect on I{kappa}B{alpha} phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. In functional studies, inhibition of I{kappa}B{alpha} phosphorylation reverses sCD40L-induced platelet activation and potentiation of platelet aggregation in response to a sub-threshold concentration of collagen. This study demonstrates that the sCD40L/CD40 axis triggers NF-{kappa}B activation in platelets. This signaling pathway plays a critical role in platelet activation and aggregation upon sCD40L stimulation and may represent an important target against thrombo

  14. Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease.

    Science.gov (United States)

    Bresson, D; Chardès, T; Chapal, N; Bès, C; Cerutti, M; Devauchelle, G; Bouanani, M; Mani, J C; Péraldi-Roux, S

    2001-01-01

    Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.

  15. Characterization and lytic activity of Pseudomonas fluorescens phages from sewage

    Directory of Open Access Journals (Sweden)

    Ananthi Radhakrishnan

    2012-03-01

    Full Text Available Pseudomonas fluorescens phages from sewage were tested against P. fluorescens isolates of soil and sewage. The phages were characterized as to host range, morphology, structural proteins and genome fingerprint. Of the seven phages isolated, one was found to be abundant in sewage (5.9×10(7 pfu/mL, having broad host range, and distinct protein and DNA profile when compared to the other six phages. DNA restriction and protein profiles of the phages and their morphology indicate the diversity in the sewage environment. None of the isolates from the rhizosphere regions of various cultivated soils were susceptible to phages isolated from sewage.

  16. Burkholderia cepacia complex Phage-Antibiotic Synergy (PAS): antibiotics stimulate lytic phage activity.

    Science.gov (United States)

    Kamal, Fatima; Dennis, Jonathan J

    2015-02-01

    The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.

  17. Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model.

    Directory of Open Access Journals (Sweden)

    Abraham Lin

    Full Text Available Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage, these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

  18. In Vivo Imaging of Molecularly Targeted Phage

    Directory of Open Access Journals (Sweden)

    Kimberly A. Kelly

    2006-12-01

    Full Text Available Rapid identification of in vivo affinity ligands would have far-reaching applications for imaging specific molecular targets, in vivo systems imaging, and medical use. We have developed a high-throughput method for identifying and optimizing ligands to map and image biologic targets of interest in vivo. We directly labeled viable phage clones with far-red fluorochromes and comparatively imaged them in vivo by multichannel fluorescence ratio imaging. Using Secreted Protein Acidic and Rich in Cysteine (osteonectin and vascular cell adhesion molecule-1 as model targets, we show that: 1 fluorescently labeled phage retains target specificity on labeling; 2 in vivo distribution can be quantitated (detection thresholds of ~ 300 phage/mm3 tissue throughout the entire depth of the tumor using fluorescent tomographic imaging; and 3 fluorescently labeled phage itself can serve as a replenishable molecular imaging agent. The described method should find widespread application in the rapid in vivo discovery and validation of affinity ligands and, importantly, in the use of fluorochrome-labeled phage clones as in vivo imaging agents.

  19. Radiolysis studies of aqueous kappa-carrageenan

    Energy Technology Data Exchange (ETDEWEB)

    Abad, L.V., E-mail: lvabad@pnri.dost.gov.p [Philippine Nuclear Research Institute, Diliman, Quezon City (Philippines); Kudo, H. [Nuclear Professional School, Graduate School of Engineering, University of Tokyo, 2-22 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1188 (Japan); Saiki, S. [Nuclear Professional School, Graduate School of Engineering, University of Tokyo, 2-22 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1188 (Japan); Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292 (Japan); Nagasawa, N.; Tamada, M. [Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292 (Japan); Fu, H.; Muroya, Y. [Nuclear Professional School, Graduate School of Engineering, University of Tokyo, 2-22 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1188 (Japan); Lin, M.; Katsumura, Y. [Nuclear Professional School, Graduate School of Engineering, University of Tokyo, 2-22 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1188 (Japan); Advanced Science Research Center, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Naka, Ibaraki 319-1195 (Japan); Relleve, L.S.; Aranilla, C.T.; DeLaRosa, A.M. [Philippine Nuclear Research Institute, Diliman, Quezon City (Philippines)

    2010-05-15

    The effects on N{sub 2}O and N{sub 2} gas on the radiation degradation yield of aqueous kappa (kappa-) carrageenan were investigated. The G{sub d} of solution saturated with N{sub 2}O solution was expectedly much higher than in air (1.7 and 1.2 x 10{sup -7} mol J{sup -1}). On the other hand, a lower G{sub d} of 1.1 x 10{sup -7} mol J{sup -1} was obtained from kappa-carrageenan solution saturated with N{sub 2}. The rate constant of reaction of OH radicals with sonicated and irradiated kappa-carrageenan were determined using e-beam pulse radiolysis. The rate constant of OH{sup c}entre dot interaction with sonicated kappa-carrageenan decreased with decreasing molecular weight. On the other hand, the OH{sup c}entre dot interaction with irradiated kappa-carrageenan decreased but did not vary significantly with decreasing molecular weight. Metal ion (Na{sup +}) induced conformational transition into helical form decreased the rate constant of OH{sup c}entre dot reaction with kappa-carrageenan. Likewise, the G{sub d} in aqueous form was affected by the conformational state of kappa-carrageenan. The helical conformation gave a lower G{sub d} (7 x 10{sup -8} mol J{sup -1}) than the coiled conformation (G{sub d} = 1.2 x 10{sup -7} mol J{sup -1}).

  20. Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

    Directory of Open Access Journals (Sweden)

    Surendra S Negi

    2009-01-01

    Full Text Available Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu, the antibody mAb Bo2C11 targeting the C2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.Availability: Users can access the EpiSearch from our web

  1. Aerosol phage therapy efficacy in Burkholderia cepacia complex respiratory infections.

    Science.gov (United States)

    Semler, Diana D; Goudie, Amanda D; Finlay, Warren H; Dennis, Jonathan J

    2014-07-01

    Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria.

  2. Phages of Listeria offer novel tools for diagnostics and biocontrol

    Directory of Open Access Journals (Sweden)

    Martin J Loessner

    2014-04-01

    Full Text Available Historically, bacteriophages infecting their hosts have perhaps been best known and even notorious for being a nuisance in dairy-fermentation processes. However, with the rapid progress in molecular microbiology and microbial ecology, a new dawn has risen for phages. This review will provide an overview on possible uses and applications of Listeria phages, including phage-typing, reporter phage for bacterial diagnostics, and use of phage as biocontrol agents for food safety. The use of phage-encoded enzymes such as endolysins for the detection and as antimicrobial will also be addressed. Desirable properties of candidate phages for biocontrol will be discussed. While emphasizing the enormous future potential for applications, we will also consider some of the intrinsic limitations dictated by both phage and bacterial ecology.

  3. Current taxonomy of phages infecting lactic acid bacteria

    Directory of Open Access Journals (Sweden)

    Jennifer eMahony

    2014-01-01

    Full Text Available Phages infecting lactic acid bacteria have been the focus of significant research attention over the past three decades. Through the isolation and characterization of hundreds of phage isolates, it has been possible to classify phages of the dairy starter and adjunct bacteria Lactococus lactis, Streptococcus thermophilus, Leuconostoc spp. and Lactobacillus spp. Among these, phages of L. lactis have been most thoroughly scrutinized and serve as an excellent model system to address issues that arise when attempting taxonomic classification of phages infecting other LAB species. Here, we present an overview of the current taxonomy of phages infecting LAB genera of industrial significance, the methods employed in these taxonomic efforts and how these may be employed for the taxonomy of phages of currently underrepresented and emerging phage species.

  4. HostPhinder: A Phage Host Prediction Tool

    DEFF Research Database (Denmark)

    Villarroel, Julia; Kleinheinz, Kortine Annina; Jurtz, Vanessa Isabell

    2016-01-01

    The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within...... reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference...... phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k) is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST...

  5. Characterization of NF-kappa B/I kappa B proteins in zebra fish and their involvement in notochord development.

    Science.gov (United States)

    Correa, Ricardo G; Tergaonkar, Vinay; Ng, Jennifer K; Dubova, Ilir; Izpisua-Belmonte, Juan Carlos; Verma, Inder M

    2004-06-01

    Although largely involved in innate and adaptive immunity, NF-kappa B plays an important role in vertebrate development. In chicks, the inactivation of the NF-kappa B pathway induces functional alterations of the apical ectodermal ridge, which mediates limb outgrowth. In mice, the complete absence of NF-kappa B activity leads to prenatal death and neural tube defects. Here, we report the cloning and characterization of NF-kappa B/I kappa B proteins in zebra fish. Despite being ubiquitously expressed among the embryonic tissues, NF-kappa B/I kappa B members present distinct patterns of gene expression during the early zebra fish development. Biochemical assays indicate that zebra fish NF-kappa B proteins are able to bind consensus DNA-binding (kappa B) sites and inhibitory I kappa B alpha proteins from mammals. We show that zebra fish I kappa B alphas are degraded in a time-dependent manner after induction of transduced murine embryo fibroblasts (MEFs) and that these proteins are able to rescue NF-kappa B activity in I kappa B alpha(-/-) MEFs. Expression of a dominant-negative form of the murine I kappa B alpha (mI kappa B alpha M), which is able to block NF-kappa B in zebra fish cells, interferes with the notochord differentiation, generating no tail (ntl)-like embryos. This phenotype can be rescued by coinjection of the T-box gene ntl (Brachyury homologue), which is typically required for the formation of posterior mesoderm and axial development, suggesting that ntl lies downstream of NF-kappa B . We further show that ntl and Brachyury promoter regions contain functional kappa B sites and NF-kappa B can directly modulate ntl expression. Our study illustrates the conservation and compatibility of NF-kappa B/I kappa B proteins among vertebrates and the importance of NF-kappa B pathway in mesoderm formation during early embryogenesis.

  6. European regulatory conundrum of phage therapy.

    Science.gov (United States)

    Verbeken, Gilbert; De Vos, Daniel; Vaneechoutte, Mario; Merabishvili, Maya; Zizi, Martin; Pirnay, Jean-Paul

    2007-10-01

    The treatment of infectious diseases with antibiotics is becoming increasingly challenging. Very few new antimicrobials are in the pharmaceutical industry pipeline. One of the potential alternatives for antibiotics is phage therapy. Major obstacles for the clinical application of bacteriophages are a false perception of viruses as 'enemies of life' and the lack of a specific frame for phage therapy in the current Medicinal Product Regulation. Short-term borderline solutions under the responsibility of a Medical Ethical Committee and/or under the umbrella of the Declaration of Helsinki are emerging. As a long-term solution, however, we suggest the creation of a specific section for phage therapy under the Advanced Therapy Medicinal Product Regulation.

  7. Phage therapy in the food industry.

    Science.gov (United States)

    Endersen, Lorraine; O'Mahony, Jim; Hill, Colin; Ross, R Paul; McAuliffe, Olivia; Coffey, Aidan

    2014-01-01

    Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future.

  8. Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Lee, Myeung Su [Division of Rheumatology, Department of Internal Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Oh, Jaemin, E-mail: jmoh@wku.ac.kr [Department of Anatomy, School of Medicine, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Institute for Skeletal Disease, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Kim, Ju-Young, E-mail: kimjy1014@gmail.com [Imaging Science-based Lung and Bone Diseases Research Center, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    2015-05-29

    Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation.

  9. TLR4 activates NF-{kappa}B in human ovarian granulosa tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Woods, Dori C., E-mail: dwoods2@partners.org [Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114 (United States); White, Yvonne A.R. [Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114 (United States); Dau, Caroline [University of California, San Francisco, School of Dentistry, San Francisco, CA 94143 (United States); Johnson, A.L. [Center for Reproductive Biology and Health, The Pennsylvania State University, University Park, PA 16802 (United States)

    2011-06-17

    Highlights: {yields} TLR4 is expressed in human ovarian granulosa tumor cells. {yields} Acting through TLR4, LPS and HSP60 induce a NF{kappa}B signaling cascade in human ovarian granulosa tumor cells. {yields} NF{kappa}B activation or inhibition did not alter chemosensitivity to TRAIL or cisplatin. -- Abstract: Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-{kappa}B) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to I{kappa}B degradation and activation of NF-{kappa}B. NF-{kappa}B activation was confirmed by nuclear localization of NF-{kappa}B p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-{kappa}B signaling attenuated LPS-induced TNF{alpha} plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-{kappa}B signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-{kappa}B does not sensitize GCTs to TRAIL or cisplatin.

  10. The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

    Directory of Open Access Journals (Sweden)

    Gill Jason J

    2012-10-01

    Full Text Available Abstract Background The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK to 280 kb encoding 448 proteins (CcrColossus, and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions Due to their lack of any apparent relationship to

  11. Vi I typing phage for generalized transduction of Salmonella typhi.

    Science.gov (United States)

    Cerquetti, M C; Hooke, A M

    1993-01-01

    Salmonella typhi Vi typing phages were used to transduce temperature-sensitive (Ts) mutants of Salmonella typhi. Antibiotic resistance and Ts+ markers were transduced at high frequency (> 10(-4) per virulent phage). Several markers were cotransduced by phage Vi I, suggesting that it may be useful for mapping studies of the S. typhi genome. PMID:8349572

  12. Phage therapy reduces Campylobacter jejuni colonization in broilers

    NARCIS (Netherlands)

    Wagenaar, J.A.; Bergen, van M.A.P.; Mueller, M.A.; Wassenaar, T.M.; Carlton, R.M.

    2005-01-01

    The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days, start

  13. HostPhinder: A Phage Host Prediction Tool

    Directory of Open Access Journals (Sweden)

    Julia Villarroel

    2016-05-01

    Full Text Available The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2].

  14. Phage therapy reduces Campylobacter jejuni colonization in broilers

    NARCIS (Netherlands)

    Wagenaar, J.A.; Bergen, van M.A.P.; Mueller, M.A.; Wassenaar, T.M.; Carlton, R.M.

    2005-01-01

    The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days,

  15. Vi I typing phage for generalized transduction of Salmonella typhi.

    OpenAIRE

    Cerquetti, M C; Hooke, A M

    1993-01-01

    Salmonella typhi Vi typing phages were used to transduce temperature-sensitive (Ts) mutants of Salmonella typhi. Antibiotic resistance and Ts+ markers were transduced at high frequency (> 10(-4) per virulent phage). Several markers were cotransduced by phage Vi I, suggesting that it may be useful for mapping studies of the S. typhi genome.

  16. Cell biology perspectives in phage biology.

    Science.gov (United States)

    Ansaldi, Mireille

    2012-01-01

    Cellular biology has long been restricted to large cellular organisms. However, as the resolution of microscopic methods increased, it became possible to study smaller cells, in particular bacterial cells. Bacteriophage biology is one aspect of bacterial cell biology that has recently gained insight from cell biology. Despite their small size, bacteriophages could be successfully labeled and their cycle studied in the host cells. This review aims to put together, although non-extensively, several cell biology studies that recently pushed the elucidation of key mechanisms in phage biology, such as the lysis-lysogeny decision in temperate phages or genome replication and transcription, one step further.

  17. Diversity and geographical distribution of Flavobacterium psychrophilum isolates and their phages: patterns of susceptibility to phage infection and phage host range.

    Science.gov (United States)

    Castillo, Daniel; Christiansen, Rói Hammershaimb; Espejo, Romilio; Middelboe, Mathias

    2014-05-01

    Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that "enhanced infection" is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.

  18. Complete genome sequence of Vibrio anguillarum phage CHOED successfully used for phage therapy in aquaculture

    DEFF Research Database (Denmark)

    Romero, Jaime; Higuera, Gastón; Gajardo, Felipe

    2014-01-01

    Vibrio anguillarum phage CHOED was isolated from Chilean mussels. It is a virulent phage showing effective inhibition of V. anguillarum. CHOED has potential in phage therapy, because it can protect fish from vibriosis in fish farms. Here, we announce the completely sequenced genome of V. anguilla...

  19. The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1

    Directory of Open Access Journals (Sweden)

    Waddell Thomas E

    2009-04-01

    Full Text Available Abstract Based upon whole genome and proteome analysis, Escherichia coli O157:H7-specific bacteriophage (phage wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage φEa21-4. The genome characteristics of phage wV8 (size 88.49 kb, mol%G+C 38.9, 138 ORFs, 23 tRNAs are very similar to those of phage Felix O1 (86.16 kb, 39.0 mol%G+C, 131 ORFs and 22 tRNAs and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein.

  20. Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria.

    Science.gov (United States)

    Alcaine, S D; Pacitto, D; Sela, D A; Nugen, S R

    2015-11-21

    Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

  1. Interaction Analysis through Proteomic Phage Display

    Directory of Open Access Journals (Sweden)

    Gustav N. Sundell

    2014-01-01

    Full Text Available Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs, or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.

  2. Recognition of epoxy with phage displayed peptides.

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2013-07-01

    The development of a general approach for non-destructive chemical and biological functionalization of epoxy could expand opportunities for both fundamental studies and creating various device platforms. Epoxy shows unique electrical, mechanical, chemical and biological compatibility and has been widely used for fabricating a variety of devices. Phage display has emerged as a powerful method for selecting peptides that possess enhanced selectivity and binding affinity toward a variety of targets. In this letter, we demonstrate for the first time a powerful yet benign approach for identifying binding motifs to epoxy via comprehensively screened phage displayed peptides. Our results show that the epoxy can be selectively recognized with peptide-displaying phages. Further, along with the development of epoxy-based microstructures; recognition of the epoxy with phage displayed peptides can be specifically localized in these microstructures. We anticipate that these results could open up exciting opportunities in the use of peptide-recognized epoxy in fundamental biochemical recognition studies, as well as in applications ranging from analytical devices, hybrid materials, surface and interface, to cell biology. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Proinflammatory effects of pancreatic elastase are mediated through TLR4 and NF-kappaB.

    Science.gov (United States)

    Hietaranta, Antti; Mustonen, Harri; Puolakkainen, Pauli; Haapiainen, Reijo; Kemppainen, Esko

    2004-10-01

    Pancreatic elastase has been implicated in the pathophysiology of severe acute pancreatitis, characterized by systemic inflammatory response, distant organ failure, and high mortality. Here we show that pancreatic elastase activates transcription factors NF-kappaB, AP-1, and NFAT in human myeloid cells (U-937 and THP-1) in culture. Pancreatic elastase also induces TNF-alpha secretion and increased expression of CD11b in THP-1 cells which can be inhibited by neutralizing anti-Toll-like receptor 4 (TLR4) antibodies. NF-kappaB blocking agents (MG-132, PGA1) prevented elastase-induced TNF-alpha secretion from THP-1 cells. Our results suggest that pancreatic elastase-induced proinflammatory effects are mediated by TLR4 and NF-kappaB in human myeloid cells.

  4. Phage-bacteria interaction network in human oral microbiome.

    Science.gov (United States)

    Wang, Jinfeng; Gao, Yuan; Zhao, Fangqing

    2016-07-01

    Although increasing knowledge suggests that bacteriophages play important roles in regulating microbial ecosystems, phage-bacteria interaction in human oral cavities remains less understood. Here we performed a metagenomic analysis to explore the composition and variation of oral dsDNA phage populations and potential phage-bacteria interaction. A total of 1,711 contigs assembled with more than 100 Gb shotgun sequencing data were annotated to 104 phages based on their best BLAST matches against the NR database. Bray-Curtis dissimilarities demonstrated that both phage and bacterial composition are highly diverse between periodontally healthy samples but show a trend towards homogenization in diseased gingivae samples. Significantly, according to the CRISPR arrays that record infection relationship between bacteria and phage, we found certain oral phages were able to invade other bacteria besides their putative bacterial hosts. These cross-infective phages were positively correlated with commensal bacteria while were negatively correlated with major periodontal pathogens, suggesting possible connection between these phages and microbial community structure in oral cavities. By characterizing phage-bacteria interaction as networks rather than exclusively pairwise predator-prey relationships, our study provides the first insight into the participation of cross-infective phages in forming human oral microbiota.

  5. How to Name and Classify Your Phage: An Informal Guide

    Directory of Open Access Journals (Sweden)

    Evelien Adriaenssens

    2017-04-01

    Full Text Available With this informal guide, we try to assist both new and experienced phage researchers through two important stages that follow phage discovery; that is, naming and classification. Providing an appropriate name for a bacteriophage is not as trivial as it sounds, and the effects might be long-lasting in databases and in official taxon names. Phage classification is the responsibility of the Bacterial and Archaeal Viruses Subcommittee (BAVS of the International Committee on the Taxonomy of Viruses (ICTV. While the BAVS aims at providing a holistic approach to phage taxonomy, for individual researchers who have isolated and sequenced a new phage, this can be a little overwhelming. We are now providing these researchers with an informal guide to phage naming and classification, taking a “bottom-up” approach from the phage isolate level.

  6. Significant differences in physicochemical properties of human immunoglobulin kappa and lambda CDR3 regions

    Directory of Open Access Journals (Sweden)

    Catherine L Townsend

    2016-09-01

    Full Text Available Antibody variable regions are composed of a heavy and a light chain and in humans there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain CDR-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 - light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains, and probed the Protein Data Bank (PDB to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda and heavy chain gene rearrangements are correlated within donors, but can differ between donors. This indicates that TdT may work with differing efficiencies between different people, but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

  7. Significant Differences in Physicochemical Properties of Human Immunoglobulin Kappa and Lambda CDR3 Regions.

    Science.gov (United States)

    Townsend, Catherine L; Laffy, Julie M J; Wu, Yu-Chang Bryan; Silva O'Hare, Joselli; Martin, Victoria; Kipling, David; Fraternali, Franca; Dunn-Walters, Deborah K

    2016-01-01

    Antibody variable regions are composed of a heavy and a light chain, and in humans, there are two light chain isotypes: kappa and lambda. Despite their importance in receptor editing, the light chain is often overlooked in the antibody literature, with the focus being on the heavy chain complementarity-determining region (CDR)-H3 region. In this paper, we set out to investigate the physicochemical and structural differences between human kappa and lambda light chain CDR regions. We constructed a dataset containing over 29,000 light chain variable region sequences from IgM-transcribing, newly formed B cells isolated from human bone marrow and peripheral blood. We also used a published human naïve dataset to investigate the CDR-H3 properties of heavy chains paired with kappa and lambda light chains and probed the Protein Data Bank to investigate the structural differences between kappa and lambda antibody CDR regions. We found that kappa and lambda light chains have very different CDR physicochemical and structural properties, whereas the heavy chains with which they are paired do not differ significantly. We also observed that the mean CDR3 N nucleotide addition in the kappa, lambda, and heavy chain gene rearrangements are correlated within donors but can differ between donors. This indicates that terminal deoxynucleotidyl transferase may work with differing efficiencies between different people but the same efficiency in the different classes of immunoglobulin chain within one person. We have observed large differences in the physicochemical and structural properties of kappa and lambda light chain CDR regions. This may reflect different roles in the humoral immune response.

  8. Twelve previously unknown phage genera are ubiquitous in global oceans.

    Science.gov (United States)

    Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

    2013-07-30

    Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

  9. $\\kappa$-Deformation and Spectral Triples

    CERN Document Server

    Iochum, B; Schücker, Th; Sitarz, A; 10.5506/APhysPolBSupp.4.305

    2011-01-01

    The aim of the paper is to answer the following question: does $\\kappa$-deformation fit into the framework of noncommutative geometry in the sense of spectral triples? Using a compactification of time, we get a discrete version of $\\kappa$-Minkowski deformation via $C^*$-algebras of groups. The dynamical system of the underlying groups (including some Baumslag--Solitar groups) is used in order to construct \\emph{finitely summable} spectral triples. This allows to bypass an obstruction to finite-summability appearing when using the common regular representation.

  10. Strong Langmuir turbulence in Kappa distributed plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Liu Sanqiu [Department of Physics and School of Materials Science and Engineering, Nanchang University, Nanchang, 330047 (China); Chen Hui [School of Materials Science and Engineering, Nanchang University, Nanchang 330047 (China)

    2012-01-15

    Superthermal electrons are often observed in space and astrophysics and can be appropriate modeled by the family of Kappa distribution functions. Taking the nonlinear wave-wave, wave-particle interactions and the effect of superthermal electrons into account, the strong Langmuir turbulence is investigated in kinetic regime. The modified Zakharov equations are obtained for the case of no damping or driving terms. On the basis of these equations, dynamics of collapse have been studied by the means of the general virial theorem, and the collapse thresholds which are strong modified by superthermal index {kappa}{sub e} are given.

  11. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  12. Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library.

    Science.gov (United States)

    Ohtani, Maki; Hikima, Jun-ichi; Jung, Tae-Sung; Kondo, Hidehiro; Hirono, Ikuo; Takeyama, Haruko; Aoki, Takashi

    2013-02-01

    Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.

  13. Studies on the adrenomedullary dependence of kappa-opioid agonist-induced diuresis in conscious rats.

    Science.gov (United States)

    Borkowski, K. R.

    1989-01-01

    1. The dependence of kappa-opioid agonist-induced diuresis, upon an intact and functional adrenal medulla in conscious rats, was investigated in order to test the hypothesis that the diuresis is mediated by a blood-borne 'diuretic factor', of adrenomedullary origin, released by kappa-opioid receptor stimulation. 2. Confirming previous observations, adrenal demedullation significantly attenuated diuretic responses to the kappa-opioid agonists U50488H, ethylketocyclazocine (EKC) and tifluadom, but did not affect basal urine output, furosemide-induced diuresis or the antidiuretic response to the mu-opioid agonist, buprenorphine. Naloxone abolished U50488H-induced diuresis, confirming an involvement of opioid receptors. 3. Transfusion studies established that blood, from intact rats treated with U50488H, induced diuresis in intact and demedullated recipient rats, whether or not the recipients had been pretreated with naloxone. However, blood from demedullated rats treated with U50448H was unable to induce diuresis when administered to intact or demedullated recipients. 4. It is concluded that kappa-opioid agonist-induced diuresis is dependent upon an intact and functional adrenal medulla and appears to be mediated by a blood-borne 'diuretic factor' of adrenomedullary origin. PMID:2558758

  14. Assembling filamentous phage occlude pIV channels.

    Science.gov (United States)

    Marciano, D K; Russel, M; Simon, S M

    2001-07-31

    Filamentous phage f1 is exported from its Escherichia coli host without killing the bacterial cell. Phage-encoded protein pIV, which is required for phage assembly and secretion, forms large highly conductive channels in the outer membrane of E. coli. It has been proposed that the phage are extruded across the bacterial outer membrane through pIV channels. To test this prediction, we developed an in vivo assay by using a mutant pIV that functions in phage export but whose channel opens in the absence of phage extrusion. In E. coli lacking its native maltooligosacharride transporter LamB, this pIV variant allowed oligosaccharide transport across the outer membrane. This entry of oligosaccharide was decreased by phage production and still further decreased by production of phage that cannot be released from the cell surface. Thus, exiting phage block the pIV-dependent entry of oligosaccharide, suggesting that phage occupy the lumen of pIV channels. This study provides the first evidence, to our knowledge, for viral exit through a large aqueous channel.

  15. Convergent evolution of pathogenicity islands in helper cos phage interference

    Science.gov (United States)

    Manning, Keith A.; Dokland, Terje; Marina, Alberto

    2016-01-01

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution. This article is part of the themed issue ‘The new bacteriology’. PMID:27672154

  16. Convergent evolution of pathogenicity islands in helper cos phage interference.

    Science.gov (United States)

    Carpena, Nuria; Manning, Keith A; Dokland, Terje; Marina, Alberto; Penadés, José R

    2016-11-05

    Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'.

  17. Precisely modulated pathogenicity island interference with late phage gene transcription.

    Science.gov (United States)

    Ram, Geeta; Chen, John; Ross, Hope F; Novick, Richard P

    2014-10-07

    Having gone to great evolutionary lengths to develop resistance to bacteriophages, bacteria have come up with resistance mechanisms directed at every aspect of the bacteriophage life cycle. Most genes involved in phage resistance are carried by plasmids and other mobile genetic elements, including bacteriophages and their relatives. A very special case of phage resistance is exhibited by the highly mobile phage satellites, staphylococcal pathogenicity islands (SaPIs), which carry and disseminate superantigen and other virulence genes. Unlike the usual phage-resistance mechanisms, the SaPI-encoded interference mechanisms are carefully crafted to ensure that a phage-infected, SaPI-containing cell will lyse, releasing the requisite crop of SaPI particles as well as a greatly diminished crop of phage particles. Previously described SaPI interference genes target phage functions that are not required for SaPI particle production and release. Here we describe a SaPI-mediated interference system that affects expression of late phage gene transcription and consequently is required for SaPI and phage. Although when cloned separately, a single SaPI gene totally blocks phage production, its activity in situ is modulated accurately by a second gene, achieving the required level of interference. The advantage for the host bacteria is that the SaPIs curb excessive phage growth while enhancing their gene transfer activity. This activity is in contrast to that of the clustered regularly interspaced short palindromic repeats (CRISPRs), which totally block phage growth at the cost of phage-mediated gene transfer. In staphylococci the SaPI strategy seems to have prevailed during evolution: The great majority of Staphylococcus aureus strains carry one or more SaPIs, whereas CRISPRs are extremely rare.

  18. Morphology, genome sequence, and structural proteome of type phage P335 from Lactococcus lactis

    DEFF Research Database (Denmark)

    Labrie, Simon J.; Josephsen, Jytte; Neve, Horst;

    2008-01-01

    Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except...

  19. Uncoupling of inflammation and insulin resistance by NF-kappaB in transgenic mice through elevated energy expenditure.

    Science.gov (United States)

    Tang, Tianyi; Zhang, Jin; Yin, Jun; Staszkiewicz, Jaroslaw; Gawronska-Kozak, Barbara; Jung, Dae Young; Ko, Hwi Jin; Ong, Helena; Kim, Jason K; Mynatt, Randy; Martin, Roy J; Keenan, Michael; Gao, Zhanguo; Ye, Jianping

    2010-02-12

    To study the metabolic activity of NF-kappaB, we investigated phenotypes of two different mouse models with elevated NF-kappaB activities. The transcriptional activity of NF-kappaB is enhanced either by overexpression of NF-kappaB p65 (RelA) in aP2-p65 mice or inactivation of NF-kappaB p50 (NF-kappaB1) through gene knock-out. In these models, energy expenditure was elevated in day and night time without a change in locomotion. The mice were resistant to adulthood obesity and diet-induced obesity without reduction in food intake. The adipose tissue growth and adipogenesis were inhibited by the elevated NF-kappaB activity. Peroxisome proliferator-activator receptor gamma expression was reduced by NF-kappaB at the transcriptional level. The two models exhibited elevated inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) in adipose tissue and serum. However, insulin sensitivity was not reduced by the inflammation in the mice on a chow diet. On a high fat diet, the mice were protected from insulin resistance. The glucose infusion rate was increased more than 30% in the hyperinsulinemic-euglycemic clamp test. Our data suggest that the transcription factor NF-kappaB promotes energy expenditure and inhibits adipose tissue growth. The two effects lead to prevention of adulthood obesity and dietary obesity. The energy expenditure may lead to disassociation of inflammation with insulin resistance. The study indicates that inflammation may prevent insulin resistance by eliminating lipid accumulation.

  20. Effect of kappa elastin on melanogenesis in A375 human melanoma cells and its related mechanism

    Institute of Scientific and Technical Information of China (English)

    TIAN Shan; HE Pei-ying; ZHANG Jian-zhong; CHEN Zhou

    2012-01-01

    Background Elastin derived peptides can regulate melanocyte precursor development.Ultraviolet irradiation,infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis.The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes.Methods A375 human melanoma cells were treated with different concentrations of kappa elastin for 24 hours.A375 human melanoma cells were randomly assigned to control,kappa elastin,and lactose pre-incubated groups.The cell viabilities were detected by the methyl thiazoleterazolium assay.Melanin content and tyrosinase activity in A375 melanoma cells were measured.The expressions of endothelin B receptor(ETBR)mRNA and c-kit mRNA in A375 melanoma cells were measured by quantative reverse transcription polymerase chain reaction.Results Fifty μg/ml of kappa elastin significantly increased the melanin content by 56.64% compared with the control(P<0.05).Kappa elastin increased cellular tyrosinase activity by 46.73% compared with the control at 24 hours(P<0.05).Kappa elastin increased the expressions of ETBR and c-kit mRNA levels by 2.13-fold and 2.47-fold compared with the controls,respectively.When pre-incubating cells with a lactose solution(10 mmol/L),the inhibition on melanin production was 34.96% compared with the kappa elastin group(P<0.05),tyrosinase activity was inhibited by 29.93% compared with kappa elastin group(P<0.05),and the expressions of ETBR mRNA and c-kit mRNA were decreased by 1.56-fold and 0.82-fold compared with kappa elastin group,respectively.Conclusion Kappa elastin increased the melanogenesis in A375 melanoma cells via the stimulation of tyrosinase activity and the expression of ETBR and c-kit.The over expressed tropoelastin produced by keratinocytes might play a role in melanogenesis of epidermal melanocytes.

  1. Phage therapy: delivering on the promise.

    Science.gov (United States)

    Harper, D R; Anderson, J; Enright, M C

    2011-07-01

    Bacteriophages are viruses that infect and, in many cases, destroy their bacterial targets. Within a few years of their initial discovery they were being investigated as therapeutic agents for infectious disease, an approach known as phage therapy. However, the nature of these exquisitely specific agents was not understood and much early use was both uninformed and unsuccessful. As a result they were replaced by chemical antibiotics once these became available. Although work on phage therapy continued (and continues) in Eastern Europe, this was not conducted to a standard allowing it to support clinical uses in areas regulated by the European Medicines Agency or the US FDA. To develop phage therapy for these areas requires work carried out in accordance with the requirements of these agencies, and, driven by the current crisis of antibiotic resistance, such clinical trials are now under way. The first Phase I clinical trial of safety was reported in 2005, and the results of the first Phase II clinical trial of efficacy of a bacteriophage therapeutic was published in 2009. While the delivery of these relatively large and complex agents to the site of disease can be more challenging than for conventional, small-molecule antibiotics, bacteriophages are then able to multiply locally even from an extremely low (picogram range) initial dose. This multiplication where and only where they are needed underlies the potential for bacteriophage therapeutics to become a much needed and powerful weapon against bacterial disease.

  2. Exploring the risks of phage application in the environment

    Directory of Open Access Journals (Sweden)

    Sean eMeaden

    2013-11-01

    Full Text Available Interest in using bacteriophages to control the growth and spread of bacterial pathogens is being revived in the wake of widespread antibiotic resistance. However, little is known about the ecological effects that high concentrations of phages in the environment might have on natural microbial communities. We review the current evidence suggesting phage-mediated environmental perturbation, with a focus on agricultural examples, and describe the potential implications for human health and agriculture. Specifically, we examine the known and potential consequences of phage application in certain agricultural practices, discuss the risks of evolved bacterial resistance to phages, and question whether the future of phage therapy will emulate that of antibiotic treatment in terms of widespread resistance. Finally, we propose some basic precautions that could preclude such phenomena and highlight existing methods for tracking bacterial resistance to phage therapeutic agents.

  3. Pitfalls to avoid when using phage display for snake toxins.

    Science.gov (United States)

    Laustsen, Andreas Hougaard; Lauridsen, Line Præst; Lomonte, Bruno; Andersen, Mikael Rørdam; Lohse, Brian

    2017-02-01

    Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions.

  4. Effects of differential modulation of mu-, delta- and kappa-opioid systems on bicuculline-induced convulsions in the mouse.

    Science.gov (United States)

    Yajima, Y; Narita, M; Takahashi-Nakano, Y; Misawa, M; Nagase, H; Mizoguchi, H; Tseng, L F; Suzuki, T

    2000-04-17

    The present study investigated the effects of micro-, delta- and kappa-opioid receptor agonists on seizures produced by blockade of gamma-aminobutyric acid (GABA)-mediated synaptic transmission in the mouse. The selective GABA(A) receptor antagonist bicuculline (1.25-3 mg/kg) given subcutaneously caused dose-dependent clonic-tonic convulsions. These convulsions were potentiated by the prototypic mu-opioid receptor agonist morphine given subcutaneously 20 min prior to a subconvulsive dose of bicuculline. The potentiation by morphine was completely reversed by pretreatment intraventricularly with the selective mu-opioid receptor antagonist beta-funaltrexamine (0.5 microgram/mouse). Pretreatment intraventricularly with the selective delta-opioid receptor agonists 2-methyl-4aalpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12, 12abeta-octahydro-quinolino[2,3,3-g]isoquinoline ((-)TAN-67) or [D-Pen(2,5)]-enkephalin (DPDPE) showed a dose-dependent increase in the incidence of convulsions. Pretreatment with naltrindole (2 mg/kg, s.c.), a selective delta-opioid receptor antagonist, abolished the enhancement of the bicuculline-induced convulsions by DPDPE. In contrast, pretreatment with the selective kappa-opioid receptor agonist U-50,488H (0.6-80 mg/kg, subcutaneously or 25-100 microgram/mouse, intraventricularly) produced a dose-dependent suppression of the bicuculline-induced convulsions. The inhibitory effect of U-50,488H was completely blocked by pretreatment subcutaneously with nor-binaltorphimine (5 mg/kg), a selective kappa-opioid receptor antagonist. This study demonstrates that activation of both mu- and delta-opioid receptors increases the incidence of convulsions produced by blockade of GABA-mediated synaptic transmission, while stimulation of kappa-opioid receptors has an anticonvulsive effect.

  5. Lysogenic Conversion and Phage Resistance Development in Phage Exposed Escherichia coli Biofilms

    Directory of Open Access Journals (Sweden)

    Abram Aertsen

    2013-01-01

    Full Text Available In this study, three-day old mature biofilms of Escherichia coli were exposed once to either a temperate Shiga-toxin encoding phage (H-19B or an obligatory lytic phage (T7, after which further dynamics in the biofilm were monitored. As such, it was found that a single dose of H-19B could rapidly lead to a near complete lysogenization of the biofilm, with a subsequent continuous release of infectious H-19B particles. On the other hand, a single dose of T7 rapidly led to resistance development in the biofilm population. Together, our data indicates a profound impact of phages on the dynamics within structured bacterial populations.

  6. Probing Tumor Microenvironment With In Vivo Phage Display

    Science.gov (United States)

    2014-10-01

    technology. A, Peptides found in the “Phage pool alone” group are listed in a descending order of frequency. Note that CISQERGESC (CIS) and CIFSGEGESC ( CIF ...expressing CISQERGESC (CIS: 0.6% of the recovered phage clones) and 202 phages expressing a relevant peptide CIFSGEGESC ( CIF : 0.2%) (Table 1A). The...term ended showed that CIS and CIF , which have very similar amino acid sequences, bind to cultured hb6011 CAFs especially to filopodia and fibrous

  7. Evolutionary Rationale for Phages as Complements of Antibiotics.

    Science.gov (United States)

    Torres-Barceló, Clara; Hochberg, Michael E

    2016-04-01

    Antibiotic-resistant bacterial infections are a major concern to public health. Phage therapy has been proposed as a promising alternative to antibiotics, but an increasing number of studies suggest that both of these antimicrobial agents in combination are more effective in controlling pathogenic bacteria than either alone. We advocate the use of phages in combination with antibiotics and present the evolutionary basis for our claim. In addition, we identify compelling challenges for the realistic application of phage-antibiotic combined therapy.

  8. DISTRIBUTION OF PHAGE TYPES AND TRANSFERABLE DRUG RESISTANCE IN SHIGELLAE

    Directory of Open Access Journals (Sweden)

    K.Badalian

    1981-08-01

    Full Text Available A total of 610 strains of Shigellae isolated from cases of diarrhea in Iran during 1962-73 were studied with respect to their phage type, as well as antibiotic resistance and transferable drug resistance along with serotyping. It was shown that there was some relation between serotypes and phage types but no association could be found between phage types and resistance pattern.

  9. Rearrangement of mouse immunoglobulin kappa deleting element recombining sequence promotes immune tolerance and lambda B cell production.

    Science.gov (United States)

    Vela, José Luis; Aït-Azzouzene, Djemel; Duong, Bao Hoa; Ota, Takayuki; Nemazee, David

    2008-02-01

    The recombining sequence (RS) of mouse and its human equivalent, the immunoglobulin (Ig) kappa deleting element (IGKDE), are sequences found at the 3' end of the Ig kappa locus (Igk) that rearrange to inactivate Igk in developing B cells. RS recombination correlates with Ig lambda (Iglambda) light (L) chain expression and likely plays a role in receptor editing by eliminating Igk genes encoding autoantibodies. A mouse strain was generated in which the recombination signal of RS was removed, blocking RS-mediated Igk inactivation. In RS mutant mice, receptor editing and self-tolerance were impaired, in some cases leading to autoantibody formation. Surprisingly, mutant mice also made fewer B cells expressing lambda chain, whereas lambda versus kappa isotype exclusion was only modestly affected. These results provide insight into the mechanism of L chain isotype exclusion and indicate that RS has a physiological role in promoting the formation of lambda L chain-expressing B cells.

  10. Bacteriophages and Phage-Derived Proteins – Application Approaches

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes – peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases – that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general. PMID:25666799

  11. Quality and safety requirements for sustainable phage therapy products.

    Science.gov (United States)

    Pirnay, Jean-Paul; Blasdel, Bob G; Bretaudeau, Laurent; Buckling, Angus; Chanishvili, Nina; Clark, Jason R; Corte-Real, Sofia; Debarbieux, Laurent; Dublanchet, Alain; De Vos, Daniel; Gabard, Jérôme; Garcia, Miguel; Goderdzishvili, Marina; Górski, Andrzej; Hardcastle, John; Huys, Isabelle; Kutter, Elizabeth; Lavigne, Rob; Merabishvili, Maia; Olchawa, Ewa; Parikka, Kaarle J; Patey, Olivier; Pouilot, Flavie; Resch, Gregory; Rohde, Christine; Scheres, Jacques; Skurnik, Mikael; Vaneechoutte, Mario; Van Parys, Luc; Verbeken, Gilbert; Zizi, Martin; Van den Eede, Guy

    2015-07-01

    The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

  12. Bacteriophages and phage-derived proteins--application approaches.

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara

    2015-01-01

    Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes - peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases - that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general.

  13. Revisiting phage therapy: new applications for old resources.

    Science.gov (United States)

    Nobrega, Franklin L; Costa, Ana Rita; Kluskens, Leon D; Azeredo, Joana

    2015-04-01

    The success of phage therapy is dependent on the development of strategies able to overcome the limitations of bacteriophages as therapeutic agents, the creation of an adequate regulatory framework, the implementation of safety protocols, and acceptance by the general public. Many approaches have been proposed to circumvent phages' intrinsic limitations but none have proved to be completely satisfactory. In this review we present the major hurdles of phage therapy and the solutions proposed to circumvent them. A thorough discussion of the advantages and drawbacks of these solutions is provided and special attention is given to the genetic modification of phages as an achievable strategy to shape bacteriophages to exhibit desirable biological properties.

  14. Isolation and Characterization of Phages Infecting Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Anna Krasowska

    2015-01-01

    Full Text Available Bacteriophages have been suggested as an alternative approach to reduce the amount of pathogens in various applications. Bacteriophages of various specificity and virulence were isolated as a means of controlling food-borne pathogens. We studied the interaction of bacteriophages with Bacillus species, which are very often persistent in industrial applications such as food production due to their antibiotic resistance and spore formation. A comparative study using electron microscopy, PFGE, and SDS-PAGE as well as determination of host range, pH and temperature resistance, adsorption rate, latent time, and phage burst size was performed on three phages of the Myoviridae family and one phage of the Siphoviridae family which infected Bacillus subtilis strains. The phages are morphologically different and characterized by icosahedral heads and contractile (SIOΦ, SUBω, and SPOσ phages or noncontractile (ARπ phage tails. The genomes of SIOΦ and SUBω are composed of 154 kb. The capsid of SIOΦ is composed of four proteins. Bacteriophages SPOσ and ARπ have genome sizes of 25 kbp and 40 kbp, respectively. Both phages as well as SUBω phage have 14 proteins in their capsids. Phages SIOΦ and SPOσ are resistant to high temperatures and to the acid (4.0 and alkaline (9.0 and 10.0 pH.

  15. Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

    2012-01-01

    The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359

  16. Learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications.

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

    2012-12-01

    The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application.

  17. Interference with phage lambda development by the small subunit of the phage 21 terminase, gp1.

    OpenAIRE

    1991-01-01

    Bacteriophage lambda development is blocked in cells carrying a plasmid that expresses the terminase genes of phage 21. The interference is caused by the small subunit of phage 21 terminase, gp1. Mutants of lambda able to form plaques in the presence of gp1 include sti mutants. One such mutation, sti30, is an A. T-to-G.C transition mutation at base pair 184 on the lambda chromosome. The sti30 mutation extends the length of the ribosome-binding sequence of the Nul gene that is complementary to...

  18. Chronic unpredictable stress exacerbates lipopolysaccharide-induced activation of nuclear factor-kappaB in the frontal cortex and hippocampus via glucocorticoid secretion.

    Science.gov (United States)

    Munhoz, Carolina Demarchi; Lepsch, Lucilia B; Kawamoto, Elisa Mitiko; Malta, Marília Brinati; Lima, Larissa de Sá; Avellar, Maria Christina Werneck; Sapolsky, Robert M; Scavone, Cristoforo

    2006-04-01

    Although the anti-inflammatory actions of glucocorticoids (GCs) are well established in the periphery, these stress hormones can increase inflammation under some circumstances in the brain. The transcription factor nuclear factor-kappaB (NF-kappaB), which is inhibited by GCs, regulates numerous genes central to inflammation. In this study, the effects of stress, GCs, and NMDA receptors on lipopolysaccharide (LPS)-induced activation of NF-kappaB in the brain were investigated. One day after chronic unpredictable stress (CUS), nonstressed and CUS rats were treated with saline or LPS and killed 2 h later. CUS potentiated the increase in LPS-induced activation of NF-kappaB in frontal cortex and hippocampus but not in the hypothalamus. This stress effect was blocked by pretreatment of rats with RU-486, an antagonist of the GC receptor. MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], an NMDA receptor antagonist, also reduced the effect of LPS in all three brain regions. However, the combined antagonism of both GC and NMDA receptors produced no further reduction in NF-kappaB activation when compared with the effect of each treatment alone. Our results indicate that stress, via GC secretion, can increase LPS-induced NF-kappaB activation in the frontal cortex and hippocampus, agreeing with a growing literature demonstrating proinflammatory effects of GCs.

  19. 1,25-dihydroxyvitamin D{sub 3} impairs NF-{kappa}B activation in human naive B cells

    Energy Technology Data Exchange (ETDEWEB)

    Geldmeyer-Hilt, Kerstin, E-mail: kerstin.hilt@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Heine, Guido, E-mail: guido.heine@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Hartmann, Bjoern, E-mail: bjoern.hartmann@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany); Baumgrass, Ria, E-mail: baumgrass@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Radbruch, Andreas, E-mail: radbruch@drfz.de [Deutsches Rheuma-Forschungszentrum Berlin, Chariteplatz 1, 10117 Berlin (Germany); Worm, Margitta, E-mail: margitta.worm@charite.de [Allergie-Centrum-Charite, CCM, Klinik fuer Dermatologie und Allergologie, Charite - Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin (Germany)

    2011-04-22

    Highlights: {yields} In naive B cells, VDR activation by calcitriol results in reduced NF-{kappa}B p105 and p50 protein expression. {yields} Ligating the VDR with calcitriol causes reduced nuclear translocation of NF-{kappa}B p65. {yields} Reduced nuclear amount of p65 after calcitriol incubation results in reduced binding of p65 on the p105 promoter. {yields} Thus, vitamin D receptor signaling may reduce or prevent activation of B cells and unwanted immune responses, e.g. in IgE dependent diseases such as allergic asthma. -- Abstract: 1{alpha},25-dihydroxyvitamin D{sub 3} (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-{kappa}B p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-{kappa}B mediated activation of human naive B cells. Naive B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human naive B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-{kappa}B activation by interference with NF-{kappa}B p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in naive B cells, namely by reducing CD40 signaling.

  20. Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.

    Directory of Open Access Journals (Sweden)

    Yihui Yuan

    Full Text Available Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs. It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.

  1. Properties of sigma and kappa Production Amplitudes

    CERN Document Server

    Ishida, M; Ishida, Muneyuki; Ishida, Shin

    2003-01-01

    Our method of analysis, which led to existence of sigma and kappa mesons, is reviewed and examined from a viewpoint of general S-matrix. It is shown that the method is consistent with the constraints from chiral symmetry and unitarity. Accordingly the long-believed common analyses of pipi (Kpi) scattering and production processes, based on elastic unitarity, prove to lose its theoretical base. The observed phase motion by 180 degrees of sigma shows also the validity of our method.

  2. Complete Genome Sequences of Four Novel Escherichia coli Bacteriophages Belonging to New Phage Groups

    DEFF Research Database (Denmark)

    Carstens, Alexander B; Kot, Witold; Hansen, Lars H

    2015-01-01

    Here, we describe the sequencing and genome annotations of a set of four Escherichia coli bacteriophages (phages) belonging to newly discovered groups previously consisting of only a single phage and thus expand our knowledge of these phage groups.......Here, we describe the sequencing and genome annotations of a set of four Escherichia coli bacteriophages (phages) belonging to newly discovered groups previously consisting of only a single phage and thus expand our knowledge of these phage groups....

  3. Differential forms and {kappa}-Minkowski spacetime from extended twist

    Energy Technology Data Exchange (ETDEWEB)

    Juric, Tajron; Meljanac, Stjepan [Rudjer Boskovic Institute, Zagreb (Croatia); Strajn, Rina [Jacobs University Bremen, Bremen (Germany)

    2013-07-15

    We analyze bicovariant differential calculus on {kappa}-Minkowski spacetime. It is shown that corresponding Lorentz generators and noncommutative coordinates compatible with bicovariant calculus cannot be realized in terms of commutative coordinates and momenta. Furthermore, {kappa}-Minkowski space and NC forms are constructed by twist related to a bicrossproduct basis. It is pointed out that the consistency condition is not satisfied. We present the construction of {kappa}-deformed coordinates and forms (super-Heisenberg algebra) using extended twist. It is compatible with bicovariant differential calculus with {kappa}-deformed igl(4)-Hopf algebra. The extended twist leading to {kappa}-Poincare-Hopf algebra is also discussed. (orig.)

  4. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii.

    Science.gov (United States)

    Hamdi, Sana; Rousseau, Geneviève M; Labrie, Simon J; Kourda, Rim S; Tremblay, Denise M; Moineau, Sylvain; Slama, Karim B

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus.

  5. NOTCH1 can initiate NF-kappaB activation via cytosolic interactions with components of the T cell signalosome

    Directory of Open Access Journals (Sweden)

    Lisa M Minter

    2014-05-01

    Full Text Available T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10 and MALT1, which is required for TCR-mediated NF-kappaB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-kappaB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-kappaB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCtheta, a T cell-specific kinase important for CBM assembly and classical NF-kappaB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCtheta and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10 and MALT1 were lost. This failure in CBM assembly reduced IkappaBalpha phosphorylation and diminished NF-kappaB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-kappaB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-kappaB signaling.

  6. The Central Reinforcing Properties of Ethanol Are Mediated by Endogenous Opioid Systems: Effects of Mu and Kappa Opioid Antagonists.

    Directory of Open Access Journals (Sweden)

    Norman E. Spear

    2009-09-01

    Full Text Available Endogenous opioid systems are implicated in the reinforcing effects of ethanol and may play a substantial role in modulating the central reinforcing effects of ethanol early in ontogeny. This possibility was explored in the present study through the use of an olfactory conditioning paradigm with centrally administered ethanol serving as an unconditioned stimulus (US. In Experiment 1, newborn rat pups were treated with either a selective mu antagonist CTOP or kappa selective antagonist nor-BNI prior to olfactory conditioning. Experiment 2 tested the effectiveness of an alternative, shorter-duration kappa opioid antagonist GNTI in altering ethanol reinforcement. Experiment 3 investigated whether the effectiveness of pharmacological blockade of opioid receptors was due to the disruption of learning per se using an olfactory aversive conditioning paradigm with intraoral quinine serving as a US. Central administration of either mu or kappa opioid antagonists prior to conditioning disrupted the reinforcing effects of ethanol in newborn rats. The kappa opioid antagonist GNTI was as effective as nor-BNI. These effects of opioid antagonists on ethanol reinforcement are unlikely to be due to a disruption of all types of conditioning, since CTOP did not affect aversive reinforcement to intraoral infusions of quinine. The present results support the hypothesis that in newborn rats, the reinforcing properties of ethanol are mediated by the endogenous activity at mu and kappa opioid receptors.

  7. Malt1 and cIAP2-Malt1 as effectors of NF-kappaB activation: kissing cousins or distant relatives?

    Science.gov (United States)

    Kingeter, Lara M; Schaefer, Brian C

    2010-01-01

    Malt1 is a multi-domain cytosolic signaling molecule that was originally identified as the target of recurrent translocations in a large fraction of MALT lymphomas. The product of this translocation is a chimeric protein in which the N-terminus is contributed by the apoptosis inhibitor, cIAP2, and the C-terminus is contributed by Malt1. Early studies suggested that Malt1 is an essential intermediate in antigen receptor activation of NF-kappaB, and that the juxtaposition of the cIAP2 N-terminus and the Malt1 C-terminus results in deregulation of Malt1 NF-kappaB stimulatory activity. Initial experimental data further suggested that the molecular mechanisms of Malt1- and cIAP-Malt1-mediated NF-kappaB activation were quite similar. However, a number of more recent studies of both Malt1 and cIAP2-Malt1 now reveal that these proteins influence NF-kappaB activation by multiple distinct mechanisms, several of which are non-overlapping. Currently available data suggest a revised model in which cIAP2-Malt1 induces NF-kappaB activation via a mechanism that depends equally on domains contributed by cIAP2 and Malt1, which confer spontaneous oligomerization activity, polyubiquitin binding, proteolytic activity, and association with and activation of TRAF2 and TRAF6 at several independent binding sites. By contrast, emerging data suggest that the wild-type Malt1 protein uniquely contributes to NF-kappaB activation primarily through the control of two proteolytic cleavage mechanisms. Firstly, Malt1 directly cleaves and inactivates A20, a negative regulator of the antigen receptor-to-NF-kappaB pathway. Secondly, Malt1 interacts with caspase-8, inducing caspase-8 cleavage of c-FLIP(L), initiating a pathway that contributes to activation of the I kappaB kinase (IKK) complex. Furthermore, data suggest that Malt1 plays a more limited and focused role in antigen receptor activation of NF-kappaB, serving to augment weak antigen signals and stimulate a defined subset of NF-kappa

  8. Computational models of populations of bacteria and lytic phage.

    Science.gov (United States)

    Krysiak-Baltyn, Konrad; Martin, Gregory J O; Stickland, Anthony D; Scales, Peter J; Gras, Sally L

    2016-11-01

    The use of phages to control and reduce numbers of unwanted bacteria can be traced back to the early 1900s, when phages were explored as a tool to treat infections before the wide scale use of antibiotics. Recently, phage therapy has received renewed interest as a method to treat multiresistant bacteria. Phages are also widely used in the food industry to prevent the growth of certain bacteria in foods, and are currently being explored as a tool for use in bioremediation and wastewater treatment. Despite the large body of biological research on phages, relatively little attention has been given to computational modeling of the population dynamics of phage and bacterial interactions. The earliest model was described by Campbell in the 1960s. Subsequent modifications to this model include partial or complete resistance, multiple phage binding sites, and spatial heterogeneity. This review provides a general introduction to modeling of the population dynamics of bacteria and phage. The review introduces the basic model and relevant concepts and evaluates more complex variations of the basic model published to date, including a model of disease epidemics caused by infectious bacteria. Finally, the shortcomings and potential ways to improve the models are discussed.

  9. Heat tolerance of dairy lactococcal c2 phages

    DEFF Research Database (Denmark)

    Nielsen, Cecilie Lykke Marvig; Basheer, Aideh; Neve, H.

    2011-01-01

    Nine Lactococcus lactis c2 phages propagated on different hosts were screened for thermal resistance in skimmed milk. Pronounced variations in thermal resistance were found. Three phages displayed high sensitivity towards heat resulting in >8 log reductions after 70 °C for 5 min, whereas the most...

  10. Fluorescent T7 display phages obtained by translational frameshift

    NARCIS (Netherlands)

    Slootweg, E.J.; Keller, H.J.H.G.; Hink, M.A.; Borst, J.W.; Bakker, J.; Schots, A.

    2006-01-01

    Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of pha

  11. Biochemical and functional interactions of a selective kappa opioid agonist with calcium

    Energy Technology Data Exchange (ETDEWEB)

    VonVoigtlander, P.F.; Ochoa, M.C.; Lewis, R.A.

    1987-01-01

    The discovery of the selective kappa opioid receptor agonist, U-50488H, has provided a tool for the study of the mechanisms and function of the kappa receptor-effector. We have investigated the interactions of this compound with calcium in several biochemical and functional studies to assess the involvement of calcium mechanisms in the kappa receptor-linked effector. In rat brain synaptosomes, U-50488H attenuated the uptake of /sup 45/Ca++ induced by K+ (40 mM) depolarization. This effect was concentration-related (U-50488H 10(-5) to 10(-7) M), was apparent in short (8-second) but not longer (1-minute) term incubations, and did not occur in the presence of a non-polarizing concentration (5.6 mM) of K+. Naloxone (10(-7) M) did not block this effect of U-50488H (10(-6) M), and higher concentrations (10(-5) M) alone blocked calcium uptake. We have found that the binding of the depolarizing amino acid analog, kainic acid, is enhanced by CaCl2. U-50488H (10(-4) to 10(-6) M) blocks this enhancement of /sup 3/H-kainic acid binding in vitro and also blocks the in vivo effects of kainic acid. In mice, intravenous injection of kainic acid causes scratching, convulsions, and death, depending on the dose administered. U-50488H blocks all of these effects (ED50 = 4.5 mg/kg for antagonism of convulsions induced by 27.5 mg/kg kainic acid). The convulsions induced by intracerebroventricularly administered kainic acid are also blocked by U-50488H as are those induced by similarly administered Bay K 8644, a calcium channel activator. All of these anticonvulsant effects of U-50488H were antagonized by naltrexone. Together these data indicate that the kappa agonist U-50488H has functionally relevant interactions with depolarization-related Ca++ mechanisms in the central nervous system.

  12. Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

    Directory of Open Access Journals (Sweden)

    Annika Gillis

    2014-07-01

    Full Text Available Many bacteriophages (phages have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here.

  13. Filamentous Phages As a Model System in Soft Matter Physics.

    Science.gov (United States)

    Dogic, Zvonimir

    2016-01-01

    Filamentous phages have unique physical properties, such as uniform particle lengths, that are not found in other model systems of rod-like colloidal particles. Consequently, suspensions of such phages provided powerful model systems that have advanced our understanding of soft matter physics in general and liquid crystals in particular. We described some of these advances. In particular we briefly summarize how suspensions of filamentous phages have provided valuable insight into the field of colloidal liquid crystals. We also describe recent experiments on filamentous phages that have elucidated a robust pathway for assembly of 2D membrane-like materials. Finally, we outline unique structural properties of filamentous phages that have so far remained largely unexplored yet have the potential to further advance soft matter physics and material science.

  14. Chemical posttranslational modification of phage-displayed peptides.

    Science.gov (United States)

    Ng, Simon; Tjhung, Katrina F; Paschal, Beth M; Noren, Christopher J; Derda, Ratmir

    2015-01-01

    Phage-displayed peptide library has fueled the discovery of novel ligands for diverse targets. A new type of phage libraries that displays not only linear and disulfide-constrained cyclic peptides but moieties that cannot be encoded genetically or incorporated easily by bacterial genetic machinery has emerged recently. Chemical posttranslational modification of phage library is one of the simplest approaches to encode nonnatural moieties. It confers the library with new functionality and makes it possible to select and evolve molecules with properties not found in the peptides, for instance, glycopeptides recognized by carbohydrate-binding protein and peptides with photoswitching capability. To this end, we describe the newly emerging techniques to chemically modify the phage library and quantify the efficiency of the reaction with a biotin-capture assay. Finally, we provide the methods to construct N-terminal Ser peptide library that allows site-selective modification of phage.

  15. Phage abortive infection in lactococci: variations on a theme.

    Science.gov (United States)

    Chopin, Marie-Christine; Chopin, Alain; Bidnenko, Elena

    2005-08-01

    Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance.

  16. Phage Particles as Vaccine Delivery Vehicles: Concepts, Applications and Prospects.

    Science.gov (United States)

    Jafari, Narjes; Abediankenari, Saeid

    2015-01-01

    The development of new strategies for vaccine delivery for generating protective and long-lasting immune responses has become an expanding field of research. In the last years, it has been recognized that bacteriophages have several potential applications in the biotechnology and medical fields because of their intrinsic advantages, such as ease of manipulation and large-scale production. Over the past two decades, bacteriophages have gained special attention as vehicles for protein/peptide or DNA vaccine delivery. In fact, whole phage particles are used as vaccine delivery vehicles to achieve the aim of enhanced immunization. In this strategy, the carried vaccine is protected from environmental damage by phage particles. In this review, phage-based vaccine categories and their development are presented in detail, with discussion of the potential of phage-based vaccines for protection against microbial diseases and cancer treatment. Also reviewed are some recent advances in the field of phage- based vaccines.

  17. Antagonist peptides of human interferon-α2b isolated from phage display library inhibit interferon induced antiviral activity

    Institute of Scientific and Technical Information of China (English)

    Wang TIAN; Gang BAI; Zheng-he LI; Wen-bo YANG

    2006-01-01

    Aim: To screen human interferon (IFN)-α2b antagonist peptides from a phage displayed heptapeptide library. Methods: WISH cells and polyclonal anti-IFN-α2b antibodies were used to select IFN receptor-binding peptides from a phage displayed heptapeptide library. The specific binding of phage clones was examined by phage ELISA and immunohistochemistry. The specific binding activities of synthetic peptides to WISH cells were detected by competition assay. Effects of synthetic peptides to IFN-induced antiviral activity were analyzed by evaluating the cytopathic effect (CPE) using the MTT method. Results: Twenty-three positive clones were obtained after seven rounds of selection. Ten clones were randomly picked from the positive clones and were sequenced. The corresponding amino acid sequences suggested 3 groups homologous to the 3 domains of IFN-α2b, defined by residues 24-41, 43-49, and 148-158 of IFN-α2b. As they presented as corresponding to IFN receptor-binding domains, AB loop and E helix, clone № 26 and 35 were chosen for further characterization and shown to bind to WISH cells. Two peptides corresponding to clone № 26 and 35, designated SP-7(SLSPGLP) and FY-7(FSAPVRY) were shown to compete with GFP-IFN-α2b for binding to its receptor and to inhibit the IFN-α2b-induced antiviral activity. Conclusion: Both IFN-α2b antagonist peptides, SP-7 and FY-7, were able to inhibit the IFN-induced antiviral activity, and could be helpful in laying the foundation for the molecular mechanism of the interaction between IFN and its receptor.

  18. The evolution of vertebrate opioid receptors

    OpenAIRE

    Stevens, Craig W.

    2009-01-01

    The proteins that mediate the analgesic and other effects of opioid drugs and endogenous opioid peptides are known as opioid receptors. Opioid receptors consist of a family of four closely-related proteins belonging to the large superfamily of G-protein coupled receptors. The three types of opioid receptors shown unequivocally to mediate analgesia in animal models are the mu (MOR), delta (DOR), and kappa (KOR) opioid receptor proteins. The role of the fourth member of the opioid receptor fami...

  19. Staphylococcus aureus phage types and their correlation to antibiotic resistance

    Directory of Open Access Journals (Sweden)

    Mehndiratta P

    2010-10-01

    Full Text Available Context: Staphylococcus aureus is one of the most devastating human pathogen. The organism has a differential ability to spread and cause outbreak of infections. Characterization of these strains is important to control the spread of infection in the hospitals as well as in the community. Aim: To identify the currently existing phage groups of Staphylococcus aureus, their prevalence and resistance to antibiotics. Materials and Methods: Study was undertaken on 252 Staphylococcus aureus strains isolated from clinical samples. Strains were phage typed and their resistance to antibiotics was determined following standard microbiological procedures. Statistical Analysis: Chi square test was used to compare the antibiotic susceptibility between methicillin resistant Staph. aureus (MRSA and methicillin sensitive S. aureus (MSSA strains. Results: Prevalence of MRSA and MSSA strains was found to be 29.36% and 70.65% respectively. Of these 17.56% of MRSA and 40.44% of MSSA strains were community acquired. All the MSSA strains belonging to phage type 81 from the community were sensitive to all the antibiotics tested including clindamycin and were resistant to penicillin. Forty five percent strains of phage group III and 39% of non-typable MRSA strains from the hospital were resistant to multiple antibiotics. Conclusion: The study revealed that predominant phage group amongst MRSA strains was phage group III and amongst MSSA from the community was phage group NA (phage type 81. MSSA strains isolated from the community differed significantly from hospital strains in their phage type and antibiotic susceptibility. A good correlation was observed between community acquired strains of phage type 81 and sensitivity to gentamycin and clindamycin.

  20. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

    Science.gov (United States)

    Holguín, Angela V.; Rangel, Guillermo; Clavijo, Viviana; Prada, Catalina; Mantilla, Marcela; Gomez, María Catalina; Kutter, Elizabeth; Taylor, Corinda; Fineran, Peter C.; Barrios, Andrés Fernando González; Vives, Martha J.

    2015-01-01

    Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%). However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization. PMID:26274971

  1. Plasma Dispersion Function for the Kappa Distribution

    Science.gov (United States)

    Podesta, John J.

    2004-01-01

    The plasma dispersion function is computed for a homogeneous isotropic plasma in which the particle velocities are distributed according to a Kappa distribution. An ordinary differential equation is derived for the plasma dispersion function and it is shown that the solution can be written in terms of Gauss' hypergeometric function. Using the extensive theory of the hypergeometric function, various mathematical properties of the plasma dispersion function are derived including symmetry relations, series expansions, integral representations, and closed form expressions for integer and half-integer values of K.

  2. The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and delivery considerations for use in Pseudomonas aeruginosa-infected airways.

    Science.gov (United States)

    Trend, Stephanie; Fonceca, Angela M; Ditcham, William G; Kicic, Anthony; Cf, Arest

    2017-07-15

    As antimicrobial-resistant microbes become increasingly common and a significant global issue, novel approaches to treating these infections particularly in those at high risk are required. This is evident in people with cystic fibrosis (CF), who suffer from chronic airway infection caused by antibiotic resistant bacteria, typically Pseudomonas aeruginosa. One option is bacteriophage (phage) therapy, which utilises the natural predation of phage viruses upon their host bacteria. This review summarises the essential and unique aspects of the phage-microbe-human lung interactions in CF that must be addressed to successfully develop and deliver phage to CF airways. The current evidence regarding phage biology, phage-bacterial interactions, potential airway immune responses to phages, previous use of phages in humans and method of phage delivery to the lung are also summarised. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  3. Dispersal and Survival of Flavobacterium psychrophilum Phages In Vivo in Rainbow Trout and In Vitro under Laboratory Conditions: Implications for Their Use in Phage Therapy

    DEFF Research Database (Denmark)

    Madsen, Lone; Bertelsen, Sif K.; Dalsgaard, Inger

    2013-01-01

    Attention has been drawn to phage therapy as an alternative approach for controlling pathogenic bacteria such as Flavobacterium psychrophilum in salmonid aquaculture, which can give rise to high mortalities, especially in rainbow trout fry. Recently, phages have been isolated with a broad host...... range and a strong lytic potential against pathogenic F. psychrophilum under experimental conditions. However, little is known about the fate of phages at environmental conditions. Here, we quantified the dispersal and fate of F. psychrophilum phages and hosts in rainbow trout fry after intraperitoneal...... injection. Both phages and bacteria were isolated from the fish organs for up to 10 days after injection, and coinjection with both bacteria and phages resulted in a longer persistence of the phage in the fish organs, than when the fish had been injected with the phages only. The occurrence of both phage...

  4. Exploration of Phage-Host Interactions in Fish Pathogen Vibrio anguillarum and Anti-Phage Defense Strategies

    DEFF Research Database (Denmark)

    Tan, Demeng

    of V. anguillarum have been isolated, indicating that antibiotic use has to be restricted and alternatives have to be developed. Lytic phages have been demonstrated to play an essential role in preventing bacterial infection. However, phages are also known to play a critical role in the evolution......The disease vibriosis is caused by the bacterial pathogen Vibrio anguillarum and results in large losses in aquaculture both in Denmark and around the world. Antibiotics have been widely used in antimicrobial prophylaxis and treatment of vibriosis. Recently, numerous multidrug-resistant strains...... of bacterial pathogenicity development. Therefore, successful application of phage therapy in the treatment of vibriosis requires a detailed understanding of phage-host interactions, especially with regards to anti-phage defense mechanisms in the host. Part I. As a first approach, 24 V. anguillarum and 13...

  5. Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation.

    Science.gov (United States)

    Oeckinghaus, Andrea; Wegener, Elmar; Welteke, Verena; Ferch, Uta; Arslan, Seda Cöl; Ruland, Jürgen; Scheidereit, Claus; Krappmann, Daniel

    2007-11-14

    Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.

  6. Kappa distributions in the presence of a potential energy

    Science.gov (United States)

    Livadiotis, George

    2016-10-01

    Classical particle systems reside at thermal equilibrium with their velocity distribution function stabilized into a Maxwell distribution. On the contrary, collisionless and correlated particle systems, such as geophysical, space, and astrophysical plasmas, are characterized by a non-Maxwellian behavior, typically described by the so-called kappa distributions, or combinations thereof. Empirical kappa distributions have become increasingly widespread across plasma physics. A breakthrough in the field came with the connection of kappa distributions to non-extensive statistical mechanics. Understanding the statistical origin of kappa distributions was the cornerstone of further theoretical developments and applications, one of which is the generalization to the phase-space kappa distributions of a Hamiltonian with non-zero potentials. We present the theory behind the phase-space kappa distributions and discuss three important applications in collisionless plasmas: (i) origin of polytropic relation; (ii) gravitational field; (iii) barometric relation (i.e., pressure vs. altitude); and (iv) plasma magnetization.

  7. Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

    OpenAIRE

    Takeda, S; Zou, Y R; Bluethmann, H; Kitamura, D; Muller, U.; Rajewsky, K

    1993-01-01

    Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replac...

  8. 20-Hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-kappaB activity by increasing IkappaB alpha levels in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Zorica Janjetovic

    Full Text Available The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1 to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-kappaB (NF-kappaB plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-kappaB, using 1,25-dihydroxycholecalciferol (calcitriol as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFkappaB DNA binding activity as well as NF-kappaB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-kappaB inhibitor protein, IkappaB alpha, in a time dependent manner, while no changes in total NF-kappaB-p65 mRNA or protein levels were observed. Another measure of NF-kappaB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IkappaB alpha was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR, 20-hydroxycholecalciferol did not affect IkappaB alpha mRNA levels, indicating that it requires VDR for its action on NF-kappaB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-kappaB. Since NF-kappaB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases.

  9. Pinitol targets nuclear factor-kappaB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis.

    Science.gov (United States)

    Sethi, Gautam; Ahn, Kwang Seok; Sung, Bokyung; Aggarwal, Bharat B

    2008-06-01

    Pinitol (3-O-methyl-chiroinositol), a component of traditional Ayurvedic medicine (talisapatra), has been shown to exhibit anti-inflammatory and antidiabetic activities through undefined mechanisms. Because the transcription factor nuclear factor-kappaB (NF-kappaB) has been linked with inflammatory diseases, including insulin resistance, we hypothesized that pinitol must mediate its effects through modulation of NF-kappaB activation pathway. We found that pinitol suppressed NF-kappaB activation induced by inflammatory stimuli and carcinogens. This suppression was not specific to cell type. Besides inducible, pinitol also abrogated constitutive NF-kappaB activation noted in most tumor cells. The suppression of NF-kappaB activation by pinitol occurred through inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Pinitol also suppressed the NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)-1, TNFR-associated death domain, TNFR-associated factor-2, transforming growth factor-beta-activated kinase-1 (TAK-1)/TAK1-binding protein-1, and IkappaBalpha kinase but not that induced by p65. The inhibition of NF-kappaB activation thereby led to down-regulation of gene products involved in inflammation (cyclooxygenase-2), proliferation (cyclin D1 and c-myc), invasion (matrix metalloproteinase-9), angiogenesis (vascular endothelial growth factor), and cell survival (cIAP1, cIAP2, X-linked inhibitor apoptosis protein, Bcl-2, and Bcl-xL). Suppression of these gene products by pinitol enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results show that pinitol inhibits the NF-kappaB activation pathway, which may explain its ability to suppress inflammatory cellular responses.

  10. Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

    Science.gov (United States)

    Le, Shuai; He, Xuesong; Tan, Yinling; Huang, Guangtao; Zhang, Lin; Lux, Renate; Shi, Wenyuan; Hu, Fuquan

    2013-01-01

    The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

  11. Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

    Directory of Open Access Journals (Sweden)

    Shuai Le

    Full Text Available The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP. Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene, which resulted in the replacement of a positively charged lysine (K by an uncharged asparagine (N. We further demonstrated that the replacement of the tail fiber gene (ORF69 of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

  12. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  13. Kappa and q Indices: Dependence on the Degrees of Freedom

    Directory of Open Access Journals (Sweden)

    George Livadiotis

    2015-04-01

    Full Text Available The kappa distributions, or their equivalent, the q-exponential distributions, are the natural generalization of the classical Boltzmann-Maxwell distributions, applied to the study of the particle populations in collisionless space plasmas. A huge step in the development of the theory of kappa distributions and their applications in space plasma physics has been achieved with the discovery that the observed kappa distributions are connected with the solid statistical background of non-extensive statistical mechanics. Now that the statistical framework has been identified, it is straightforward to improve our understanding of the nature of the kappa index (or the entropic q-index that governs these distributions. One critical topic is the dependence of the kappa index on the degrees of freedom. In this paper, we first show how this specific dependence is naturally emerged, using the formalism of the N-particle kappa distribution of velocities. Then, the result is extended in the presence of potential energies. It is shown that the kappa index is simply related to the kinetic and potential degrees of freedom. In addition, it is shown that various problems of non-extensive statistical mechanics, such as (i the correlation dependence on the total number of particles; and (ii the normalization divergence for finite kappa indices, are resolved considering the kappa index dependence on the degrees of freedom.

  14. Kappa-Deformations: Historical Developments and Recent Results

    CERN Document Server

    Lukierski, Jerzy

    2016-01-01

    I shall recall in historical perspective some results from nineties and show further how $\\kappa$-deformed symmetries and $\\kappa$-Minkowski space inspired DSR (Doubly of Deformed Special Relativity) approach proposed after 2000. As very recent development I shall show how to describe quantum-covariant $\\kappa$-deformed phase spaces by passing from Hopf algebras to Hopf algebroids (arXiv:1507.02612) and I will briefly describe the $\\kappa$-deformations of $AdS_5 \\times S^5$ superstring target spaces (arXiv:1510.030.83).

  15. Evolution of Lactococcus lactis phages within a cheese factory.

    Science.gov (United States)

    Rousseau, Geneviève M; Moineau, Sylvain

    2009-08-01

    We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3' overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.

  16. Safety and efficacy of phage therapy via the intravenous route.

    Science.gov (United States)

    Speck, Peter; Smithyman, Anthony

    2016-02-01

    Increasing development of antimicrobial resistance is driving a resurgence in interest in phage therapy: the use of bacteriophages to treat bacterial infections. As the lytic action of bacteriophages is unaffected by the antibiotic resistance status of their bacterial target, it is thought that phage therapy may have considerable potential in the treatment of a wide range of topical and localized infections. As yet this interest has not extended to intravenous (IV) use, which is surprising given that the historical record shows that phages are likely to be safe and effective when delivered by this route. Starting almost 100 years ago, phages were administered intravenously in treatment of systemic infections including typhoid, and Staphylococcal bacteremia. There was extensive IV use of phages in the 1940s to treat typhoid, reportedly with outstanding efficacy and safety. The safety of IV phage administration is also underpinned by the detailed work of Ochs and colleagues in Seattle who have over four decades' experience with IV injection into human subjects of large doses of highly purified coliphage PhiX174. Though these subjects included a large number of immune-deficient children, no serious side effects were observed over this extended time period. The large and continuing global health problems of typhoid and Staphylococcus aureus are exacerbated by the increasing antibiotic resistance of these pathogens. We contend that these infections are excellent candidates for use of IV phage therapy.

  17. Oligopeptide M13 Phage Display in Pathogen Research

    Directory of Open Access Journals (Sweden)

    Michael Hust

    2013-10-01

    Full Text Available Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

  18. A novel fluorescent probe: europium complex hybridized T7 phage.

    Science.gov (United States)

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  19. Biofilm control with natural and genetically-modified phages.

    Science.gov (United States)

    Motlagh, Amir Mohaghegh; Bhattacharjee, Ananda Shankar; Goel, Ramesh

    2016-04-01

    Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.

  20. Coevolution of CRISPR bacteria and phage in 2 dimensions

    Science.gov (United States)

    Han, Pu; Deem, Michael

    2014-03-01

    CRISPR (cluster regularly interspaced short palindromic repeats) is a newly discovered adaptive, heritable immune system of prokaryotes. It can prevent infection of prokaryotes by phage. Most bacteria and almost all archae have CRISPR. The CRISPR system incorporates short nucleotide sequences from viruses. These incorporated sequences provide a historical record of the host and predator coevolution. We simulate the coevolution of bacteria and phage in 2 dimensions. Each phage has multiple proto-spacers that the bacteria can incorporate. Each bacterium can store multiple spacers in its CRISPR. Phages can escape recognition by the CRISPR system via point mutation or recombination. We will discuss the different evolutionary consequences of point mutation or recombination on the coevolution of bacteria and phage. We will also discuss an intriguing ``dynamic phase transition'' in the number of phage as a function of time and mutation rate. We will show that due to the arm race between phages and bacteria, the frequency of spacers and proto-spacers in a population can oscillate quite rapidly.

  1. Significance of phage-host interactions for biocontrol of Campylobacter jejuni in food

    DEFF Research Database (Denmark)

    Athina, Zampara; Sørensen, Martine Camilla Holst; Elsser-Gravesen, Anne

    2017-01-01

    Poultry meat is the main source of Campylobacter jejuni foodborne disease. Currently, no effective control measures prevent C. jejuni from contaminating poultry meat. However, post-harvest phage treatment is a promising biocontrol strategy that has not yet been explored. Here we identified phages...... capable of reducing C. jejuni at chilled temperature by a systematic screening of unique phages of our collection consisting of flagellotropic phages and phages dependent on capsular polysaccharides (CPSs) for infection. Interestingly, CPS phages showed varied killing efficiencies at 5 °C in vitro......, ranging from insignificant reduction to 0.55 log reduction. In contrast, none of the flagellotropic phages significantly reduced C. jejuni counts at low temperature. Phage adsorption at 5 °C showed that flagellotropic phages bind reversibly and less efficiently to C. jejuni than CPS phages, which may...

  2. Nuclear IL-33 is a transcriptional regulator of NF-{kappa}B p65 and induces endothelial cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yeon-Sook; Park, Jeong Ae; Kim, Jihye; Rho, Seung-Sik; Park, Hyojin [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Young-Myeong [Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon (Korea, Republic of); Kwon, Young-Guen, E-mail: ygkwon@yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer IL-33 as nuclear factor regulated expression of ICAM-1 and VCAM-1. Black-Right-Pointing-Pointer Nuclear IL-33 increased the transcription of NF-{kappa}B p65 by binding to the p65 promoter. Black-Right-Pointing-Pointer Nuclear IL-33 controls NF-{kappa}B-dependent inflammatory responses. -- Abstract: Interleukin (IL)-33, an IL-1 family member, acts as an extracellular cytokine by binding its cognate receptor, ST2. IL-33 is also a chromatin-binding transcriptional regulator highly expressed in the nuclei of endothelial cells. However, the function of IL-33 as a nuclear factor is poorly defined. Here, we show that IL-33 is a novel transcriptional regulator of the p65 subunit of the NF-{kappa}B complex and is involved in endothelial cell activation. Quantitative reverse transcriptase PCR and Western blot analyses indicated that IL-33 mediates the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in endothelial cells basally and in response to tumor necrosis factor-{alpha}-treatment. IL-33-induced ICAM-1/VCAM-1 expression was dependent on the regulatory effect of IL-33 on the nuclear factor (NF)-{kappa}B pathway; NF-{kappa}B p65 expression was enhanced by IL-33 overexpression and, conversely, reduced by IL-33 knockdown. Moreover, NF-{kappa}B p65 promoter activity and chromatin immunoprecipitation analysis revealed that IL-33 binds to the p65 promoter region in the nucleus. Our data provide the first evidence that IL-33 in the nucleus of endothelial cells participates in inflammatory reactions as a transcriptional regulator of NF-{kappa}B p65.

  3. Identification of small molecule binding sites within proteins using phage display technology.

    Energy Technology Data Exchange (ETDEWEB)

    Rodi, D. J.; Agoston, G. E.; Manon, R.; Lapcevich, R.; Green, S. J.; Makowski, L.; Biosciences Division; EntreMed Inc.; Florida State Univ.

    2001-11-01

    Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

  4. A sensitive and simple plaque formation method for the Stx2 phage of Escherichia coli O157:H7, which does not form plaques in the standard plating procedure.

    Science.gov (United States)

    Islam, Md Rakibul; Ogura, Yoshitoshi; Asadulghani, Md; Ooka, Tadasuke; Murase, Kazunori; Gotoh, Yasuhiro; Hayashi, Tetsuya

    2012-05-01

    Bacteriophages are fascinating genetic elements that play key roles in the evolution and diversification of bacterial genomes. Shiga toxin (Stx)-transducing phages are important genetic elements that disseminate the stx genes among enterohemorrhagic Escherichia coli (EHEC). They are generally regarded as lambda-like phages, but their biological and genetic properties have not been fully elucidated. This is partly due to a serious obstacle in obtaining visible plaques. Here, we describe a modified double agar overlay method that allows us to easily detect and accurately enumerate plaques of Sp5, the Stx2 phage of the EHEC O157 Sakai strain, which otherwise does not produce plaques in the standard plating procedure. In the modified method, the top agar was supplemented with mitomycin C (MMC) and Ca(2+) (or Mg(2+)). MMC appears to prevent the lysogenization of Sp5 and/or compel Sp5 to follow the lytic cycle by inducing the SOS response in the host cells. The divalent cations significantly improve phage adsorption to the host cells and thus yield a synergistic effect in combination with MMC. We further applied this method to a receptor analysis of Sp5 and obtained findings that suggest that the YaeT (BamA) protein serves as the receptor of Sp5. This method would be a very useful tool in studies of Stx2 phages and studies of other phages from various bacteria, in which researchers often encounter problems with plaque formation.

  5. Complete genome sequence of Klebsiella pneumoniae phage JD001.

    Science.gov (United States)

    Cui, Zelin; Shen, Wenbin; Wang, Zheng; Zhang, Haotian; Me, Rao; Wang, Yanchun; Zeng, Lingbin; Zhu, Yongzhang; Qin, Jinhong; He, Ping; Guo, Xiaokui

    2012-12-01

    Klebsiella pneumoniae is a member of the family Enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. The emergence of multidrug-resistant strains of K. pneumoniae has became a public health problem globally. To develop an effective antimicrobial agent, we isolated a bacteriophage, named JD001, from seawater and sequenced its genome. Comparative genome analysis of phage JD001 with other K. pneumoniae bacteriophages revealed that phage JD001 has little similarity to previously published K. pneumoniae phages KP15, KP32, KP34, and phiKO2. Here we announce the complete genome sequence of JD001 and report major findings from the genomic analysis.

  6. Phage approved in food, why not as a therapeutic?

    Science.gov (United States)

    Sarhan, Wessam A; Azzazy, Hassan M E

    2015-01-01

    Bacterial resistance is not only restricted to human infections but is also a major problem in food. With the marked decrease in produced antimicrobials, the world is now reassessing bacteriophages. In 2006, ListShield™ received the US FDA approval for using phage in food. Nevertheless, regulatory approval of phage-based therapeutics is still facing many challenges. This review highlights the use of bacteriophages as biocontrol agents in the food industry. It also focuses on the challenges still facing the regulatory approval of phage-based therapeutics and the proposed approaches to overcome such challenges.

  7. Phage display library screening for identification of interacting protein partners.

    Science.gov (United States)

    Addepalli, Balasubrahmanyam; Rao, Suryadevara; Hunt, Arthur G

    2015-01-01

    Phage display is a versatile high-throughput screening method employed to understand and improve the chemical biology, be it production of human monoclonal antibodies or identification of interacting protein partners. A majority of cell proteins operate in a concerted fashion either by stable or transient interactions. Such interactions can be mediated by recognition of small amino acid sequence motifs on the protein surface. Phage display can play a crucial role in identification of such motifs. This report describes the use of phage display for the identification of high affinity sequence motifs that could be responsible for interactions with a target (bait) protein.

  8. Review: phage therapy: a modern tool to control bacterial infections.

    Science.gov (United States)

    Qadir, Muhammad Imran

    2015-01-01

    The evolution of antibiotic-resistant in bacteria has aggravated curiosity in development of alternative therapy to conventional drugs. One of the emerging drugs that can be used alternative to antibiotics is bacteriophage therapy. The use of living phages in the cure of lethal infectious life threatening diseases caused by Gram positive and Gram negative bacteria has been reported. Another development in the field of bacteriophage therapy is the use of genetically modified and non replicating phages in the treatment of bacterial infection. Genetically engineered bacteriophages can be used as adjuvant along with antibiotic therapy. Phages encoded with lysosomal enzymes are also effectual in the treatment of infectious diseases.

  9. Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the 'functional progesterone withdrawal'.

    Science.gov (United States)

    Allport, V C; Pieber, D; Slater, D M; Newton, R; White, J O; Bennett, P R

    2001-06-01

    Human labour is associated with the up-regulation of prostaglandins within the uterus, synthesized via the type-2 cyclo-oxygenase enzyme (COX-2). These lead to remodelling of the fetal membranes and cervix and to stimulation of myometrial contractions. In the human, the principal source of prostaglandins is the amnion. Progesterone acts to promote myometrial quiescence, and in many species the onset of labour is preceded by withdrawal of progesterone. Humans show no systemic progesterone withdrawal, although biochemical changes within the uterus are similar to those in other species. A mutual negative interaction between the transcription factor nuclear factor (NF)-kappaB and the progesterone receptor (PR) has been reported. Using transient transfections and assays for transcriptional activation and promoter binding, we have shown that there is constitutive activity of NF-kappaB in amnion cells at the time of labour, and that COX-2 expression depends upon NF-kappaB. In cells obtained before labour, in which NF-kappaB activity is low, increasing the concentration of PR represses NF-kappaB dependent transcription, while stimulation with IL-1beta both increases NF-kappaB activity and represses PR activity. Our data suggest that human labour is associated with constitutive NF-kappaB activity within the amnion, which functions to increase the expression of COX-2 and appears to contribute to the 'functional progesterone withdrawal'.

  10. CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4.

    Directory of Open Access Journals (Sweden)

    Stephanie J Yaung

    Full Text Available Bacteria rely on two known DNA-level defenses against their bacteriophage predators: restriction-modification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-associated (Cas systems. Certain phages have evolved countermeasures that are known to block endonucleases. For example, phage T4 not only adds hydroxymethyl groups to all of its cytosines, but also glucosylates them, a strategy that defeats almost all restriction enzymes. We sought to determine whether these DNA modifications can similarly impede CRISPR-based defenses. In a bioinformatics search, we found naturally occurring CRISPR spacers that potentially target phages known to modify their DNA. Experimentally, we show that the Cas9 nuclease from the Type II CRISPR system of Streptococcus pyogenes can overcome a variety of DNA modifications in Escherichia coli. The levels of Cas9-mediated phage resistance to bacteriophage T4 and the mutant phage T4 gt, which contains hydroxymethylated but not glucosylated cytosines, were comparable to phages with unmodified cytosines, T7 and the T4-like phage RB49. Our results demonstrate that Cas9 is not impeded by N6-methyladenine, 5-methylcytosine, 5-hydroxymethylated cytosine, or glucosylated 5-hydroxymethylated cytosine.

  11. The phage-related chromosomal islands of Gram-positive bacteria

    OpenAIRE

    Novick, Richard P.; Christie, Gail E.; Penadés, Jose R.

    2010-01-01

    The phage-related chromosomal islands (PRCIs) were first identified in Staphylococcus aureus as highly mobile, superantigen-encoding genetic elements known as the S. aureus pathogenicity islands (SaPIs). These elements are characterized by a specific set of phage-related functions that enable them to use the phage reproduction cycle for their own transduction and inhibit phage reproduction in the process. SaPIs produce many phage-like infectious particles; their streptococcal counterparts hav...

  12. Lethal effects of /sup 32/P decay on transfecting activity of Bacillus subtillis phage phie DNA

    Energy Technology Data Exchange (ETDEWEB)

    Loveday, K.S.

    1979-07-15

    Disintegration of /sup 32/P present in the DNA of Bacillus subtilis phage phie (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only 1/12 of the /sup 32/P disintegrations per phage DNA equivalent inactivities the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by phie DNA.

  13. Coffee and Maillard products activate NF-kappaB in macrophages via H2O2 production.

    Science.gov (United States)

    Muscat, Sonja; Pelka, Joana; Hegele, Jörg; Weigle, Bernd; Münch, Gerald; Pischetsrieder, Monika

    2007-05-01

    In this study, we investigated the immunomodulatory activity of coffee and Maillard reaction products on macrophages in vitro. Stimulation of macrophages with coffee, but not with raw coffee extract in PBS, led to a 13-fold increased nuclear NF-kappaB translocation. A Maillard reaction mixture (25 mM D-ribose/L-lysine, 30 min at 120 degrees C) increased NF-kappaB translocation 18-fold (in PBS) or six-fold (in medium). MRPs also induced a two-fold increased NF-kappaB translocation in untransfected human embryonic kidney (HEK) cells as well as in HEK cells stably transfected with the receptor for advanced glycation endproducts (RAGE), indicating that the effect was not RAGE mediated. On the other hand, catalase totally abolished coffee- and MRP-induced NF-kappaB translocation. Consequently, up to 366 microM hydrogen peroxide was measured in the coffee preparation and Maillard mixtures used for cell stimulation. Stimulation of macrophages with MRPs did not lead to significantly increased IL-6 or NO release. Thus, it can be concluded that coffee and MRPs induce NF-kappaB translocation in macrophages via the generation of hydrogen peroxide.

  14. A role for NRAGE in NF-kappaB activation through the non-canonical BMP pathway.

    Science.gov (United States)

    Matluk, Nicholas; Rochira, Jennifer A; Karaczyn, Aldona; Adams, Tamara; Verdi, Joseph M

    2010-01-25

    Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-kappaB has yet to be explored. Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -alpha/beta and subsequent transcriptional activation of the p65 subunit of NF-kappaB. Ablation of endogenous NRAGE by siRNA inhibited NF-kappaB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-kappaB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor. Modulation of NRAGE expression revealed novel roles in regulating NF-kappaB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway.

  15. NF-kappaB Signaling in Chronic Inflammatory Airway Disease

    Directory of Open Access Journals (Sweden)

    Michael Schuliga

    2015-06-01

    Full Text Available Asthma and chronic obstructive pulmonary disease (COPD are obstructive airway disorders which differ in their underlying causes and phenotypes but overlap in patterns of pharmacological treatments. In both asthma and COPD, oxidative stress contributes to airway inflammation by inducing inflammatory gene expression. The redox-sensitive transcription factor, nuclear factor (NF-kappaB (NF-κB, is an important participant in a broad spectrum of inflammatory networks that regulate cytokine activity in airway pathology. The anti-inflammatory actions of glucocorticoids (GCs, a mainstay treatment for asthma, involve inhibition of NF-κB induced gene transcription. Ligand bound GC receptors (GRs bind NF-κB to suppress the transcription of NF-κB responsive genes (i.e., transrepression. However, in severe asthma and COPD, the transrepression of NF-κB by GCs is negated as a consequence of post-translational changes to GR and histones involved in chromatin remodeling. Therapeutics which target NF-κB activation, including inhibitors of IκB kinases (IKKs are potential treatments for asthma and COPD. Furthermore, reversing GR/histone acetylation shows promise as a strategy to treat steroid refractory airway disease by augmenting NF-κB transrepression. This review examines NF-κB signaling in airway inflammation and its potential as target for treatment of asthma and COPD.

  16. Genetic evidence for the involvement of the S-layer protein gene sap and the sporulation genes spo0A, spo0B, and spo0F in Phage AP50c infection of Bacillus anthracis.

    Science.gov (United States)

    Plaut, Roger D; Beaber, John W; Zemansky, Jason; Kaur, Ajinder P; George, Matroner; Biswas, Biswajit; Henry, Matthew; Bishop-Lilly, Kimberly A; Mokashi, Vishwesh; Hannah, Ryan M; Pope, Robert K; Read, Timothy D; Stibitz, Scott; Calendar, Richard; Sozhamannan, Shanmuga

    2014-03-01

    In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.

  17. Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach.

    Science.gov (United States)

    Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K

    2015-01-01

    Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

  18. Upregulation of the kappa opioidergic system in left ventricular rat myocardium in response to volume overload: Adaptive changes of the cardiac kappa opioid system in heart failure.

    Science.gov (United States)

    Treskatsch, Sascha; Shaqura, Mohammed; Dehe, Lukas; Feldheiser, Aarne; Roepke, Torsten K; Shakibaei, Mehdi; Spies, Claudia D; Schäfer, Michael; Mousa, Shaaban A

    2015-12-01

    Opioids have long been known for their analgesic effects and are therefore widely used in anesthesia and intensive care medicine. However, in the last decade research has focused on the opioidergic influence on cardiovascular function. This project thus aimed to detect the precise cellular localization of kappa opioid receptors (KOR) in left ventricular cardiomyocytes and to investigate putative changes in KOR and its endogenous ligand precursor peptide prodynorphin (PDYN) in response to heart failure. After IRB approval, heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. All rats of the control and ACF group were characterized by their morphometrics and hemodynamics. In addition, the existence and localization as well as adaptive changes of KOR and PDYN were investigated using radioligand binding, double immunofluorescence confocal analysis, RT-PCR and Western blot. Similar to the brain and spinal cord, [(3)H]U-69593 KOR selective binding sites were detected the left ventricle (LV). KOR colocalized with Cav1.2 of the outer plasma membrane and invaginated T-tubules and intracellular with the ryanodine receptor of the sarcoplasmatic reticulum. Interestingly, KOR could also be detected in mitochondria of rat LV cardiomyocytes. As a consequence of heart failure, KOR and PDYN were up-regulated on the mRNA and protein level in the LV. These findings suggest that the cardiac kappa opioidergic system might modulate rat cardiomyocyte function during heart failure.

  19. Women in Leadership Roles in Phi Delta Kappa.

    Science.gov (United States)

    Harder, Martha B.; And Others

    The professional and personal characteristics of the men and women in Phi Delta Kappa who hold the top elected positions in local chapters were compared, and the perceptions of these leaders of the impact of allowing women to be members of Phi Delta Kappa were assessed. A 43-item questionnaire was developed and mailed to 1,340 chapter presidents…

  20. The scalar kappa from D+ -> K- pi+ pi+: Further Studies

    CERN Document Server

    Göbel, C

    2003-01-01

    We briefly review the recent results obtained by Fermilab experiment E791 on the Dalitz plot analysis of the decay D+ -> K- pi+ pi+, where indication for a light Kpi scalar resonance, the kappa, was found. We also present preliminary studies providing further information on the phase behavior of the scalar components at low mass, supporting the previous indication for the kappa.

  1. Origins and properties of kappa distributions in space plasmas

    Science.gov (United States)

    Livadiotis, George

    2016-07-01

    Classical particle systems reside at thermal equilibrium with their velocity distribution function stabilized into a Maxwell distribution. On the contrary, collisionless and correlated particle systems, such as the space and astrophysical plasmas, are characterized by a non-Maxwellian behavior, typically described by the so-called kappa distributions. Empirical kappa distributions have become increasingly widespread across space and plasma physics. However, a breakthrough in the field came with the connection of kappa distributions to the solid statistical framework of Tsallis non-extensive statistical mechanics. Understanding the statistical origin of kappa distributions was the cornerstone of further theoretical developments and applications, some of which will be presented in this talk: (i) The physical meaning of thermal parameters, e.g., temperature and kappa index; (ii) the multi-particle description of kappa distributions; (iii) the phase-space kappa distribution of a Hamiltonian with non-zero potential; (iv) the Sackur-Tetrode entropy for kappa distributions, and (v) the new quantization constant, h _{*}˜10 ^{-22} Js.

  2. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  3. High-throughput Identification of Phage-derived Imaging Agents

    Directory of Open Access Journals (Sweden)

    Kimberly A. Kelly

    2006-01-01

    Full Text Available The use of phage-displayed peptide libraries is a powerful method for selecting peptides with desired binding properties. However, the validation and prioritization of “hits” obtained from this screening approach remains challenging. Here, we describe the development and testing of a new analysis method to identify and display hits from phage-display experiments and high-throughput enzyme-linked immunosorbent assay screens. We test the method using a phage screen against activated macrophages to develop imaging agents with higher specificity for active disease processes. The new methodology should be useful in identifying phage hits and is extendable to other library screening methods such as small-molecule and nanoparticle libraries.

  4. [Inactivation of T4 phage in water environment using proteinase].

    Science.gov (United States)

    Lü, Wen-zhou; Yang, Qing-xiang; Zhang, Yu; Yang, Min; Zhu, Chun-fang

    2004-09-01

    The inactivation effectiveness of proteinase to viruses was investigated by using T4 phage as a model virus. The results showed that the inactivation effectiveness of proteinase to T4 phage was obvious. In the optimum conditions and 67.5 u/mL concentration, the inactivation rate of proteinase K to T4 phage in sterilized water and in sewage achieved 99.4% and 49.4% respectively in an hour, and achieved >99.9% and 81.1% in three hours. The inactivation rate of the industrial proteinase 1398 to T4 phage in sterilized water achieved 74.4% in an hour. The effects of pH and temperature on the inactivation effectiveness was not evident.

  5. Persistence of bacteria and phages in a chemostat.

    Science.gov (United States)

    Smith, Hal L; Thieme, Horst R

    2012-05-01

    The model of bacteriophage predation on bacteria in a chemostat formulated by Levin et al. (Am Nat 111:3-24, 1977) is generalized to include a distributed latent period, distributed viral progeny release from infected bacteria, unproductive adsorption of phages to infected cells, and possible nutrient uptake by infected cells. Indeed, two formulations of the model are given: a system of delay differential equations with infinite delay, and a more general infection-age model that leads to a system of integro-differential equations. It is shown that the bacteria persist, and sharp conditions for persistence and extinction of phages are determined by the reproductive ratio for phage relative to the phage-free equilibrium. A novel feature of our analysis is the use of the Laplace transform.

  6. Complete Genome Sequence of the Streptomyces Phage Nanodon

    Science.gov (United States)

    2016-01-01

    Streptomyces phage Nanodon is a temperate double-stranded DNA Siphoviridae belonging to cluster BD1. It was isolated from soil collected in Kilauea, HI, using Streptomyces griseus subsp. griseus as a host.

  7. Inhibition of phage infection in capsule-producing Streptococcus ...

    African Journals Online (AJOL)

    SERVER

    2007-10-04

    Oct 4, 2007 ... Acid production by capsule-producing Streptococcus thermophilus was inhibited less ... lactic acid bacteria (LAB) are of similar size to fat globules ..... Characterization of new virulent phage (MLC-A) of Lactobacillus paracasei.

  8. Deep sequencing analysis of phage libraries using Illumina platform.

    Science.gov (United States)

    Matochko, Wadim L; Chu, Kiki; Jin, Bingjie; Lee, Sam W; Whitesides, George M; Derda, Ratmir

    2012-09-01

    This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve

  9. The hallucinogen derived from Salvia divinorum, salvinorin A, has kappa-opioid agonist discriminative stimulus effects in rats.

    Science.gov (United States)

    Willmore-Fordham, Catherine B; Krall, Daniel M; McCurdy, Christopher R; Kinder, David H

    2007-09-01

    Data from clinical and preclinical studies converge implicating the plant-derived hallucinogen salvinorin A as an important pharmacologic tool; this psychoactive compound may expand scientific understandings on mammalian kappa-opioid receptor systems. Human salvinorin A effects, consistent with kappa-opioid receptor agonism, include antinociception, sedation, dysphoria and distorted perceptions. The experiments reported here measured salvinorin A (1-3mg/kg, i.p.) discriminative stimulus properties in male Sprague-Dawley rats conditioned to recognize the discriminative stimulus cue generated by the well characterized kappa-opioid agonist U-69593 (0.56 mg/kg, i.p.). At three distinct active doses, salvinorin A fully substituted for U-69593 without altering response rates. The lever choice pattern in U-69593 trained animals reverted to vehicle lever responding when a kappa selective antagonist compound, nor-BNI (4.5 nM, i.c.v.) was administered 1h prior to salvinorin A, yet nor-BNI alone failed to impact the rate or pattern of subject responses. These findings confirm and extend results published after similar drug discrimination tests were performed in rhesus monkeys. The discussion section of this article highlights public concern over salvinorin A misuse and emphasizes several potential pharmacotherapeutic applications for salvinorin A or analogue compounds.

  10. Small-angle neutron scattering study on irradiated kappa carrageenan

    Energy Technology Data Exchange (ETDEWEB)

    Abad, Lucille [Neutron Science Laboratory, Institute for Solid State Physics, University of Tokyo, 106-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan) and Philippine Nuclear Research Institute, Commonwealth Ave., Diliman, Quezon City (Philippines) and Advanced Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirane Shirakata, Tokai, Ibaraki 319-1195 (Japan)]. E-mail: lvabad@pnri.dost.gov.ph; Okabe, Satoshi [Neutron Science Laboratory, Institute for Solid State Physics, University of Tokyo, 106-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan); Koizumi, Satoshi [Advanced Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirane Shirakata, Tokai, Ibaraki 319-1195 (Japan); Shibayama, Mitsuhiro [Neutron Science Laboratory, Institute for Solid State Physics, University of Tokyo, 106-1 Shirakata, Tokai, Ibaraki 319-1106 (Japan)]. E-mail: sibayama@issp.u-tokyo.ac.jp

    2006-05-31

    The structure of gamma-ray-irradiated {kappa}-carrageenan in aqueous solutions was investigated in terms of small-angle neutron scattering. The scattered intensity, I(q), of non-irradiated {kappa}-carrageenan solutions (5 wt%) was well described with an Ornstein-Zernike (OZ)-type function with the correlation length of 85 A, indicating that the {kappa}-carrageenan solution behaves just as a polymer solution in the semi-dilute regime. By increasing the irradiation dose (100 kGy), I(q) changed to a power-law function with the scattering exponent of -1.84. Further increase in dose results in a recovery of OZ-type function. This indicates that a progressive cleavage of {kappa}-carrageenan chains takes place randomly, leading to a self-similar structure at 100 kGy. This is followed by further segmentation of {kappa}-carrageenan chains.

  11. Properties of Klebsiella Phage P13 and Associated Exopolysaccharide Depolymerase

    Institute of Scientific and Technical Information of China (English)

    LIU Yang; LI Guiyang; MO Zhaolan; CHAI Zihan; SHANG Anqi; MOU Haijin

    2014-01-01

    The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide (EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of de-polymerase approached the maximum 60 min after infection. Treatment at 70℃for 30 min inactivated all the phage, but retained over 90%of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60℃and pH 6.5. Transmission electron mi-croscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a dou-ble-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation in-cluding thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.

  12. Antibody Phage Library Screening Efficiency Measured by KD Values

    Institute of Scientific and Technical Information of China (English)

    WANG Hui-tang; SHAN Ya-ming; TANG Li-li; GAO Li-zeng; WANG Li-ping; LI Wei; LI Yu-xin

    2005-01-01

    An antibody phage library was screened with two target molecules, IFNα-2a and FGFR-GST, and the KD value of each round of panning was measured. It was found that the apparent KD values decreased along with each additional panning round, which indicates the increase of the binding affinity between the phage and the target molecules.This result shows that the KD value is a reliable intrinsic parameter and a new method for screening efficiency detection is thus provided.

  13. AGRO100 inhibits activation of nuclear factor-kappaB (NF-kappaB) by forming a complex with NF-kappaB essential modulator (NEMO) and nucleolin.

    Science.gov (United States)

    Girvan, Allicia C; Teng, Yun; Casson, Lavona K; Thomas, Shelia D; Jüliger, Simone; Ball, Mark W; Klein, Jon B; Pierce, William M; Barve, Shirish S; Bates, Paula J

    2006-07-01

    AGRO100, also known as AS1411, is an experimental anticancer drug that recently entered human clinical trials. It is a member of a novel class of antiproliferative agents known as G-rich oligonucleotides (GRO), which are non-antisense, guanosine-rich phosphodiester oligodeoxynucleotides that form stable G-quadruplex structures. The biological activity of GROs results from their binding to specific cellular proteins as aptamers. One important target protein of GROs has been previously identified as nucleolin, a multifunctional protein expressed at high levels by cancer cells. Here, we report that AGRO100 also associates with nuclear factor-kappaB (NF-kappaB) essential modulator (NEMO), which is a regulatory subunit of the inhibitor of kappaB (IkappaB) kinase (IKK) complex, and also called IKKgamma. In the classic NF-kappaB pathway, the IKK complex is required for phosphorylation of IkappaBalpha and subsequent activation of the transcription factor NF-kappaB. We found that treatment of cancer cells with AGRO100 inhibits IKK activity and reduces phosphorylation of IkappaBalpha in response to tumor necrosis factor-alpha stimulation. Using a reporter gene assay, we showed that AGRO100 blocks both tumor necrosis factor-alpha-induced and constitutive NF-kappaB activity in human cancer cell lines derived from cervical, prostate, breast, and lung carcinomas. In addition, we showed that, in AGRO100-treated cancer cells, NEMO is coprecipitated by nucleolin, indicating that both proteins are present in the same complex. Our studies suggest that abrogation of NF-kappaB activity may contribute to the anticancer effects of AGRO100 and that nucleolin may play a previously unknown role in regulating the NF-kappaB pathway.

  14. New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its Escherichia coli receptor FhuA

    OpenAIRE

    Flayhan, Ali; Wien, Frank; Paternostre, Maïté; Boulanger, Pascale; Breyton, Cécile

    2012-01-01

    International audience; The majority of bacterial viruses are bacteriophages bearing a tail that serves to recognise the bacterial surface and deliver the genome into the host cell. Infection is initiated by the irreversible interaction between the viral receptor binding protein (RBP) and a receptor at the surface of the bacterium. This interaction results ultimately in the phage DNA release in the host cytoplasm. Phage T5 infects Escher-ichia coli after binding of its RBP pb5 to the outer me...

  15. DNA heats up : Energetics of genome ejection from phage revealed by isothermal titration calorimetry

    CERN Document Server

    Jeembaeva, Meerim; Castelnovo, Martin; Evilevitch, Alex

    2010-01-01

    Most bacteriophages are known to inject their double-stranded DNA into bacteria upon receptor binding in an essentially spontaneous way. This downhill thermodynamic process from the intact virion toward the empty viral capsid plus released DNA is made possible by the energy stored during active packaging of the genome into the capsid. Only indirect measurements of this energy have been available until now using either single-molecule or osmotic suppression techniques. In this paper, we describe for the first time the use of isothermal titration calorimetry to directly measure the heat released (or equivalently the enthalpy) during DNA ejection from phage lambda, triggered in solution by a solubilized receptor. Quantitative analyses of the results lead to the identification of thermodynamic determinants associated with DNA ejection. The values obtained were found to be consistent with those previously predicted by analytical models and numerical simulations. Moreover, the results confirm the role of DNA hydrat...

  16. TRAF2-binding BIR1 domain of c-IAP2/MALT1 fusion protein is essential for activation of NF-kappaB.

    Science.gov (United States)

    Garrison, J B; Samuel, T; Reed, J C

    2009-04-02

    Marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphoma is the most common extranodal non-Hodgkin lymphoma. The t(11;18)(q21;q21) translocation occurs frequently in MALT lymphomas and creates a chimeric NF-kappaB-activating protein containing the baculoviral IAP repeat (BIR) domains of c-IAP2 (inhibitor of apoptosis protein 2) fused with portions of the MALT1 protein. The BIR1 domain of c-IAP2 interacts directly with TRAF2 (TNFalpha-receptor-associated factor-2), but its role in NF-kappaB activation is still unclear. Here, we investigated the role of TRAF2 in c-IAP2/MALT1-induced NF-kappaB activation. We show the BIR1 domain of c-IAP2 is essential for NF-kappaB activation, whereas BIR2 and BIR3 domains are not. Studies of c-IAP2/MALT1 BIR1 mutant (E47A/R48A) that fails to activate NF-kappaB showed loss of TRAF2 binding, but retention of TRAF6 binding, suggesting that interaction of c-IAP2/MALT1 with TRAF6 is insufficient for NF-kappaB induction. In addition, a dominant-negative TRAF2 mutant or downregulation of TRAF2 achieved by small interfering RNA inhibited NF-kappaB activation by c-IAP2/MALT1 showing that TRAF2 is indispensable. Comparisons of the bioactivity of intact c-IAP2/MALT1 oncoprotein and BIR1 E47A/R48A c-IAP2/MALT1 mutant that cannot bind TRAF2 in a lymphoid cell line provided evidence that TRAF2 interaction is critical for c-IAP2/MALT1-mediated increases in the NF-kappaB activity, increased expression of endogenous NF-kappaB target genes (c-FLIP, TRAF1), and resistance to apoptosis.

  17. Comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25 reveals a novel siphovirus group related to phages infecting hosts of different taxonomic classes.

    Science.gov (United States)

    Flores, Víctor; Sepúlveda-Robles, Omar; Cazares, Adrián; Kameyama, Luis; Guarneros, Gabriel

    2017-08-01

    Bacteriophages (phages) are estimated to be the most abundant and diverse entities in the biosphere harboring vast amounts of novel genetic information. Despite the genetic diversity observed, many phages share common features, such as virion morphology, genome size and organization, and can readily be associated with clearly defined phage groups. However, other phages display unique genomes or, alternatively, mosaic genomes composed of regions that share homology with those of phages of diverse origins; thus, their relationships cannot be easily assessed. In this work, we present a functional and comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25, a virulent member of the Siphoviridae family. The genomes of PaMx25 and a highly homologous phage NP1, bore sequence homology and synteny with the genomes of phages that infect hosts different than Pseudomonas. In order to understand the relationship of the PaMx25 genome with that of other phages, we employed several computational approaches. We found that PaMx25 and NP1 effectively bridged several phage groups. It is expected that as more phage genomes become available, more gaps will be filled, blurring the boundaries that currently separate phage groups.

  18. Paving a regulatory pathway for phage therapy: Europe should muster the resources to financially, technically and legally support the introduction of phage therapy

    OpenAIRE

    Huys, Isabelle; Pirnay, Jean-Paul; Lavigne, Rob; Jennes, Serge; De Vos, Daniel; Casteels, Minne; Verbeken, Gilbert

    2013-01-01

    The growing problem of antibiotic-resistant bacteria has re-kindled interest in phage-based therapies. Yet, the use of phages to treat life-threatening bacterial infections is held back by the lack of an appropriate regulatory framework for phage therapy.

  19. The Legacy of 20th Century Phage Research.

    Science.gov (United States)

    Campbell, Allan M

    2010-09-01

    The Golden Age of Phage Research, where phage was the favored material for attacking many basic questions in molecular biology, lasted from about 1940 to 1970. The era was initiated by Ellis and Delbrück, whose analysis defined the relevant parameters to measure in studying phage growth, and depended on the fact that the contents of a plaque can comprise descendants of a single infecting particle. It ended around 1970 because definitive methods had then become available for answering the same questions in other systems. Some of the accomplishments of phage research were the demonstration by Hershey and Chase that the genetic material of phage T2 is largely composed of DNA, the construction of linkage maps of T2 and T4 by Hershey and Rotman and their extension to very short molecular distances by Benzer, and the isolation of conditionally lethal mutants in T4 by Epstein et al. and in λ by Campbell. The dissection of the phage life cycle into causal chains was explored by Edgar and Wood for T4 assembly and later in the regulation of lysogeny by Kaiser, extended to the molecular level by Ptashne and others. Restriction/modification was discovered in λ by Bertani and Weigle, and the biochemical mechanism was elucidated by Arber and by Smith.

  20. Phage therapy against Enterococcus faecalis in dental root canals

    Directory of Open Access Journals (Sweden)

    Leron Khalifa

    2016-09-01

    Full Text Available Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages. Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals.

  1. Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery

    Directory of Open Access Journals (Sweden)

    Teerapong Yata

    2014-01-01

    Full Text Available Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage, viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

  2. Phage therapy against Enterococcus faecalis in dental root canals

    Science.gov (United States)

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals. PMID:27640530

  3. Conserved termini and adjacent variable region of Twortlikevirus Staphylococcus phages

    Institute of Scientific and Technical Information of China (English)

    Xianglilan Zhang; Huaixing Kang; Yuyuan Li; Xiaodong Liu; Yu Yang; Shasha Li; Guangqian Pei; Qiang Sun; Peng Shu; Zhiqiang Mi; Yong Huang; Zhiyi Zhang; Yannan Liu; Xiaoping An; Xiaolu Xu; Yigang Tong

    2015-01-01

    Methicillin-resistant Staphylococcus aureus(MRSA) is an increasing cause of serious infection,both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection are narrowing because of the limited number of newly developed antimicrobials. Here, four newly-isolated MRSA-virulent phages, IME-SA1, IMESA2, IME-SA118 and IME-SA119, were sequenced and analyzed. Their genome termini were identified using our previously proposed "termini analysis theory". We provide evidence that remarkable conserved terminus sequences are found in IME-SA1/2/118/119, and, moreover, are widespread throughout Twortlikevirus Staphylococcus phage G1 and K species. Results also suggested that each phage of the two species has conserved 5′ terminus while the 3′ terminus is variable. More importantly, a variable region with a specific pattern was found to be present near the conserved terminus of Twortlikevirus S. phage G1 species. The clone with the longest variable region had variable terminus lengths in successive generations, while the clones with the shortest variable region and with the average length variable region maintained the same terminal length as themselves during successive generations. IME-SA1 bacterial infection experiments showed that the variation is not derived from adaptation of the phage to different host strains. This is the first study of the conserved terminus and variable region of Twortlikevirus S. phages.

  4. Effects of surface functionalization on the surface phage coverage and the subsequent performance of phage-immobilized magnetoelastic biosensors.

    Science.gov (United States)

    Horikawa, Shin; Bedi, Deepa; Li, Suiqiong; Shen, Wen; Huang, Shichu; Chen, I-Hsuan; Chai, Yating; Auad, Maria L; Bozack, Michael J; Barbaree, James M; Petrenko, Valery A; Chin, Bryan A

    2011-01-15

    One of the important applications for which phage-immobilized magnetoelastic (ME) biosensors are being developed is the wireless, on-site detection of pathogenic bacteria for food safety and bio-security. Until now, such biosensors have been constructed by immobilizing a landscape phage probe on gold-coated ME resonators via physical adsorption. Although the physical adsorption method is simple, the immobilization stability and surface coverage of phage probes on differently functionalized sensor surfaces need to be evaluated as a potential way to enhance the detection capabilities of the biosensors. As a model study, a filamentous fd-tet phage that specifically binds streptavidin was adsorbed on either bare or surface-functionalized gold-coated ME resonators. The surface functionalization was performed through the formation of three self-assembled monolayers with a different terminator, based on the sulfur-gold chemistry: AC (activated carboxy-terminated), ALD (aldehyde-terminated), and MT (methyl-terminated). The results, obtained by atomic force microscopy, showed that surface functionalization has a large effect on the surface phage coverage (46.8%, 49.4%, 4.2%, and 5.2% for bare, AC-, ALD-, and MT-functionalized resonators, respectively). In addition, a direct correlation of the observed surface phage coverage with the quantity of subsequently captured streptavidin-coated microbeads was found by scanning electron microscopy and by resonance frequency measurements of the biosensors. The differences in surface phage coverage on the differently functionalized surfaces may then be used to pattern the phage probe layer onto desired parts of the sensor surface to enhance the detection capabilities of ME biosensors.

  5. Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy.

    Science.gov (United States)

    Khan Mirzaei, Mohammadali; Nilsson, Anders S

    2015-01-01

    Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.

  6. Comparative Omics and Trait Analyses of Marine Pseudoalteromonas Phages Advance the Phage OTU Concept

    Directory of Open Access Journals (Sweden)

    Melissa B. Duhaime

    2017-07-01

    Full Text Available Viruses influence the ecology and evolutionary trajectory of microbial communities. Yet our understanding of their roles in ecosystems is limited by the paucity of model systems available for hypothesis generation and testing. Further, virology is limited by the lack of a broadly accepted conceptual framework to classify viral diversity into evolutionary and ecologically cohesive units. Here, we introduce genomes, structural proteomes, and quantitative host range data for eight Pseudoalteromonas phages isolated from Helgoland (North Sea, Germany and use these data to advance a genome-based viral operational taxonomic unit (OTU definition. These viruses represent five new genera and inform 498 unaffiliated or unannotated protein clusters (PCs from global virus metagenomes. In a comparison of previously sequenced Pseudoalteromonas phage isolates (n = 7 and predicted prophages (n = 31, the eight phages are unique. They share a genus with only one other isolate, Pseudoalteromonas podophage RIO-1 (East Sea, South Korea and two Pseudoalteromonas prophages. Mass-spectrometry of purified viral particles identified 12–20 structural proteins per phage. When combined with 3-D structural predictions, these data led to the functional characterization of five previously unidentified major capsid proteins. Protein functional predictions revealed mechanisms for hijacking host metabolism and resources. Further, they uncovered a hybrid sipho-myovirus that encodes genes for Mu-like infection rarely described in ocean systems. Finally, we used these data to evaluate a recently introduced definition for virus populations that requires members of the same population to have >95% average nucleotide identity across at least 80% of their genes. Using physiological traits and genomics, we proposed a conceptual model for a viral OTU definition that captures evolutionarily cohesive and ecologically distinct units. In this trait-based framework, sensitive hosts are

  7. B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

    OpenAIRE

    J. Chen(Florida State University, Tallahassee, U.S.A.); Trounstine, M; Kurahara, C.; Young, F.; Kuo, C C; Y. Xu; Loring, J.F.; Alt, F W; Huszar, D

    1993-01-01

    We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, ...

  8. Nanoscale bacteriophage biosensors beyond phage display.

    Science.gov (United States)

    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

  9. Nanoscale bacteriophage biosensors beyond phage display

    Directory of Open Access Journals (Sweden)

    Lee JW

    2013-10-01

    Full Text Available Jong-Wook Lee,1 Jangwon Song,1,2 Mintai P Hwang,1 Kwan Hyi Lee1,2 1Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea; 2Department of Biomedical Engineering, University of Science and Technology, Seoul, Korea Abstract: Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. Keywords: biosensing, M13 bacteriophage, T4 bacteriophage, bacterial detection, Escherichia coli, SPR sensor

  10. Nanoscale bacteriophage biosensors beyond phage display

    Science.gov (United States)

    Lee, Jong-Wook; Song, Jangwon; Hwang, Mintai P; Lee, Kwan Hyi

    2013-01-01

    Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology. PMID:24143096

  11. Four Escherichia coli O157:H7 phages: a new bacteriophage genus and taxonomic classification of T1-like phages.

    Science.gov (United States)

    Niu, Yan D; McAllister, Tim A; Nash, John H E; Kropinski, Andrew M; Stanford, Kim

    2014-01-01

    The T1-like bacteriophages vB_EcoS_AHP24, AHS24, AHP42 and AKS96 of the family Siphoviridae were shown to lyse common phage types of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7), but not non-O157 E. coli. All contained circularly permuted genomes of 45.7-46.8 kb (43.8-44 mol% G+C) encoding 74-81 open reading frames and 1 arginyl-tRNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the structural proteins were identical among the four phages. Further proteomic analysis identified seven structural proteins responsible for tail fiber, tail tape measure protein, major capsid, portal protein as well as major and minor tail proteins. Bioinformatic analyses on the proteins revealed that genomes of AHP24, AHS24, AHP42 and AKS96 did not encode for bacterial virulence factors, integration-related proteins or antibiotic resistance determinants. All four phages were highly lytic to STEC O157:H7 with considerable potential as biocontrol agents. Comparative genomic, proteomic and phylogenetic analysis suggested that the four phages along with 17 T1-like phage genomes from database of National Center for Biotechnology Information (NCBI) can be assigned into a proposed subfamily "Tunavirinae" with further classification into five genera, namely "Tlslikevirus" (TLS, FSL SP-126), "Kp36likevirus" (KP36, F20), Tunalikevirus (T1, ADB-2 and Shf1), "Rtplikevirus" (RTP, vB_EcoS_ACG-M12) and "Jk06likevirus" (JK06, vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49). The fact that the viruses related to JK06 have been isolated independently in Israel (JK06) (GenBank Assession #, NC_007291), Canada (vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96) and Mexico (phiKP26, phiJLA23) (between 2005 and 2011) indicates that these similar phages are widely distributed, and that horizontal gene transfer does not always prevent the characterization of bacteriophage evolution. With this new scheme, any new discovered phages with same type can be

  12. Four Escherichia coli O157:H7 phages: a new bacteriophage genus and taxonomic classification of T1-like phages.

    Directory of Open Access Journals (Sweden)

    Yan D Niu

    Full Text Available The T1-like bacteriophages vB_EcoS_AHP24, AHS24, AHP42 and AKS96 of the family Siphoviridae were shown to lyse common phage types of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7, but not non-O157 E. coli. All contained circularly permuted genomes of 45.7-46.8 kb (43.8-44 mol% G+C encoding 74-81 open reading frames and 1 arginyl-tRNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the structural proteins were identical among the four phages. Further proteomic analysis identified seven structural proteins responsible for tail fiber, tail tape measure protein, major capsid, portal protein as well as major and minor tail proteins. Bioinformatic analyses on the proteins revealed that genomes of AHP24, AHS24, AHP42 and AKS96 did not encode for bacterial virulence factors, integration-related proteins or antibiotic resistance determinants. All four phages were highly lytic to STEC O157:H7 with considerable potential as biocontrol agents. Comparative genomic, proteomic and phylogenetic analysis suggested that the four phages along with 17 T1-like phage genomes from database of National Center for Biotechnology Information (NCBI can be assigned into a proposed subfamily "Tunavirinae" with further classification into five genera, namely "Tlslikevirus" (TLS, FSL SP-126, "Kp36likevirus" (KP36, F20, Tunalikevirus (T1, ADB-2 and Shf1, "Rtplikevirus" (RTP, vB_EcoS_ACG-M12 and "Jk06likevirus" (JK06, vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49. The fact that the viruses related to JK06 have been isolated independently in Israel (JK06 (GenBank Assession #, NC_007291, Canada (vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96 and Mexico (phiKP26, phiJLA23 (between 2005 and 2011 indicates that these similar phages are widely distributed, and that horizontal gene transfer does not always prevent the characterization of bacteriophage evolution. With this new scheme, any new discovered phages with same type