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  • Intracellular survival of virulent Bordetella pertussis in human polymorphonuclear leukocytes. 1919361

    Little is known regarding the interaction of Bordetella pertussis with polymorphonuclear leukocytes (PMNL) or the role PMNL play as an initial line of defense against B. pertussis infection. An in vitro system was developed to establish conditions for the study of phagocytosis and killing of virulent B. pertussis by human PMNL. Phagocytosis of B. pertussis strains BP504, BP165, and BP338 occurred by opsonization with anti-B. pertussis antibody, while autologous normal human sera did not induce significant phagocytosis. In PMNL bacterial killing assays virulent B. pertussis strains survived PMNL bactericidal activities while Escherichia coli controls were readily killed. Electron microscopy studies using acid phosphatase as a lysosomal marker strongly suggested that B. pertussis inhibits phagosome-lysosome fusion in PMNL. These results indicate that virulent B. pertussis strains are capable of surviving intracellularly within PMNL and that such survival may be due to inhibition of phagosome-lysosome fusion.
    Document Type:
    Reference
    Product Catalog Number:
    20-176
    Product Catalog Name:
    100X GTPγS, 10mM

  • Role of carbohydrate recognition domains of pertussis toxin in adherence of Bordetella pertussis to human macrophages. 1353482

    Pertussis toxin (PT) and filamentous hemagglutinin can each mediate the association of Bordetella pertussis with human macrophages. Adherence via filamentous hemagglutinin leads to integrin-mediated entry and survival of the bacteria within the human cell. We determined the contribution of PT to bacterial adherence to human macrophages. Plating macrophages on wells coated with recombinant PT subunit 2 (S2) or S3 decreased PT-dependent bacterial binding by greater than 60%; S1, S4, and S5 were ineffective. S3-dependent adherence was reduced 63% +/- 8% by sialic acid, while S2-dependent adherence was reduced 53% +/- 11% by galactose. Loss of the carbohydrate recognition properties of S2 by deletion of residues 40 to 54 or site-specific mutations at Asn-93, His-47, or Arg-50 eliminated the ability of the subunit protein to competitively inhibit bacterial binding. Peptides corresponding to residues 28 to 45 of S2 and S3 competitively inhibited adherence. Treatment of macrophages with antibodies to Le(a) or Le(x) but not CD14, CD15, CD18, or HLA interfered with PT-mediated binding. Exposure of the macrophages to the B oligomer, S2, or S3 increased binding to the CD11b/CD18 integrin. These results indicate that the carbohydrate recognition domains of both S2 and S3 participate in adherence of B. pertussis to human macrophages. The PT receptor(s), as yet unidentified, appears to carry the Le(a) or Le(x) determinants and is functionally capable of modulating integrin-mediated binding to the macrophage.
    Document Type:
    Reference
    Product Catalog Number:
    20-111
    Product Catalog Name:
    Assay Dilution Buffer II (ADBII)

  • Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling ... 22919671

    The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric G(q), G(i), and G(12/13) proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G(12/13) signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of G(q) and G(12/13) in mediating PMT effects on Rho/ROCK and Notch1 signaling.
    Document Type:
    Reference
    Product Catalog Number:
    AB3511

  • Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. 6438083

    Membranes from bovine brain bind relatively large quantities of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinity. The two proteins responsible for most of this activity were purified; they account for 1.5% of the membrane protein. The two proteins contain alpha subunits of either 39,000 or 41,000 Da, beta subunits of 36,000 or 35,000 Da, and a potential gamma subunit (11,000 Da). These structures are the same as a family of proteins that includes transducin and the regulatory proteins, GS and GI, of adenylate cyclase. The 41,000- and 39,000-Da polypeptides can be ADP-ribosylated with islet-activating protein from Bordetella pertussis, bind guanine nucleotides specifically, and migrate through polyacrylamide gels with rates similar to the alpha subunits of GI and transducin, respectively. The 36,000- and 35,000-Da polypeptides are similar to the beta subunits of GI and GS. The gamma subunit is found whenever beta subunits are present. The 41,000- and 39,000-Da polypeptides (with beta and gamma) are designated, respectively, GI and GO from brain. The alpha subunit of GO was isolated without the use of ligands known to dissociate other G proteins. GO alpha binds GTP gamma S reversibly in the absence of Mg2+ and is relatively stable in cholate. This isolated alpha subunit should be of great utility in elucidating the mechanism of action of this family of GTP-binding proteins.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3075
    Product Catalog Name:
    Anti-G Protein Giα-1 Antibody, clone R4

  • OmpA-mediated rickettsial adherence to and invasion of human endothelial cells is dependent upon interaction with α2β1 integrin. 23145974

    Rickettsia conorii, a member of the spotted fever group (SFG) of the genus Rickettsia and causative agent of Mediterranean spotted fever, is an obligate intracellular pathogen capable of infecting various mammalian cell types. SFG rickettsiae express two major immunodominant surface cell antigen (Sca) proteins, OmpB (Sca5) and OmpA (Sca0). While OmpB-mediated entry has been characterized, the contribution of OmpA has not been well defined. Here we show OmpA expression in Escherichia coli is sufficient to mediate adherence to and invasion of non-phagocytic human endothelial cells. A recombinant soluble C-terminal OmpA protein domain (954-1735) with predicted structural homology to the Bordetella pertussis pertactin protein binds mammalian cells and perturbs R. conorii invasion by interacting with several mammalian proteins including β1 integrin. Using functional blocking antibodies, small interfering RNA transfection, and mouse embryonic fibroblast cell lines, we illustrate the contribution of α2β1 integrin as a mammalian ligand involved in R. conorii invasion of primary endothelial cells. We further demonstrate that OmpA-mediated attachment to mammalian cells is in part dependent on a conserved non-continuous RGD motif present in a predicted C-terminal 'pertactin' domain in OmpA.Our results demonstrate that multiple adhesin-receptor pairs are sufficient in mediating efficient bacterial invasion of R. conorii.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Formaldehyde-inactivated human enterovirus 71 vaccine is compatible for co-immunization with a commercial pentavalent vaccine. 21315698

    In this study we tested the effectiveness of a formaldehyde-inactivated EV71 vaccine and its compatibility for co-immunization with a pentavalent vaccine that contained inactivated poliovirus (PV) vaccine. The inactivated EV71 vaccine (C2 genogroup) elicited an antibody response which broadly neutralized homologous and heterologous genogroups, including B4, C4, and B5. Pups from vaccinated dams were resistant to the EV71 challenge and had a high survival rate and a low tissue viral burden when compared to those from non-vaccinated counterparts. Co-immunization with pentavalent and inactivated EV71 vaccines elicited antibodies against the major components of the pentavalent vaccine including the PV, Bordetella pertussis, Haemophilus influenzae type b, diphtheria toxoid, and tetanus toxoid at the same levels as in mice immunized with pentavalent vaccine alone. Likewise, EV71 neutralizing antibody titers were comparable between EV71-vaccinated mice and mice co-immunized with the two vaccines. These results indicate that formaldehyde-inactivated whole virus EV71 vaccine is feasible for designing multivalent vaccines.Copyright © 2011 Elsevier Ltd. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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