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Berlin Brandenburg

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  • 1
    Language: English
    In: Virus Genes, 1995, Vol.10(2), pp.173-177
    Description: We describe a method for construction of hymeric bacteriophage T4 particles displaying foreign polypeptides on their surface. The method is based on our finding that minor T4 fibrous protein fibritin encoded by gene wac (whisker's antigen control) could be lengthened at the C terminus without impairing its folding or binding to the phage particle. The lengthened fibritin gene could easily be transferred into the T4 genome by homologous recombination with a plasmid containing the modified gene wac . The modified gene wac is expressed properly during phage reproduction, and the lengthened fibritin is bound to phage particles. As an example of this type of method, we have obtained the hymeric T4 particles carrying a polypeptide of 53 residues, 45 of which are from the pre-S2 region of hepatitis B virus. The T4 display vector extends currently available display systems.
    Keywords: bacteriophage T4 ; fibritin ; surface display vector ; homologous recombination ; HBV
    ISSN: 0920-8569
    E-ISSN: 1572-994X
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  • 2
    In: Proceedings of the National Academy of Sciences of the United States, Dec 19, 1995, Vol.92(26), p.12362(5)
    Description: Reverse genetics has shown that not all parental genome segments in intertypic reassortants of the three reovirus serotypes are wild type. Some reassortants have more ST1 than ST3 genome segments. In these, the ST1 is a wild type but the ST3 genome segments have mutations that make it more homologous to the segments that they replace. In reassortants produced by the crossing of ST2 and ST3, all ST2 segments are wild type but the ST3 S4 genome has two mutations that function as genome segment assortment signals.
    Keywords: Reoviruses -- Genetic Aspects ; Genomes -- Research
    ISSN: 0027-8424
    E-ISSN: 10916490
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  • 3
    In: Infection and Immunity, 2010, Vol. 78(9), p.3763
    Description: Haemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyi class I strain 35000HP (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAI dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HPhgbAI and 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbAI but not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAII from nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAI but not those challenged with strain 35000HPhgbAII. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbAI/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbAI/Freund's vaccine; however, anti-nHgbAI from both studies bound whole cells of 35000HPhgbAI better than 35000HPhgbAII and partially blocked hemoglobin binding to nHgbAI. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbAI/Freund's, the nHgbAI/MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.
    Keywords: Hemoglobin ; Hemoglobin Receptors ; Monophosphoryl Lipid A ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Heme ; Chancroid ; Vaccines ; Freund'S Adjuvant ; Immunization ; Haemophilus Ducreyi ; Microorganisms & Parasites ; Immunology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 4
    In: Infection and Immunity, 2009, Vol. 77(7), p.3065
    Description: HgbA is the sole TonB-dependent receptor for hemoglobin (Hb) acquisition of Haemophilus ducreyi. Binding of Hb to HgbA is the initial step in heme acquisition from Hb. To better understand this step, we mutagenized hgbA by deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in trans in host strain H. ducreyi FX547 hgbA. All mutant proteins were expressed, exported, and detected on the surface by anti-HgbA immunoglobulin G (IgG). Deletion of sequences in loops 5 and 7 of HgbA abolished Hb binding in two different formats. In contrast, HgbA proteins containing deletions in the other nine loops retained the ability to bind Hb. None of the clones expressing mutant proteins were able to grow on plates containing low concentrations of Hb. Previously we demonstrated in a swine model of chancroid infection that an HgbA vaccine conferred complete protection from a challenge infection. Using anti-HgbA IgG from this study and the above deletion mutants, we show that loops 4, 5, and 7 of HgbA were immunogenic and surface exposed and that IgG directed against loops 4 and 5 blocked Hb binding. Furthermore, loop 6 was cleaved by protease on intact H. ducreyi, suggesting surface exposure. These data implicate a central domain of HgbA (in respect to the primary amino acid sequence) as important in Hb binding and suggest that this region of the molecule might have potential as a subunit vaccine.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 5
    In: Infection and Immunity, 2008, Vol. 76(4), p.1608
    Description: The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 6
    In: Infection and Immunity, 2005, Vol. 73(6), p.3431
    Description: Previously, we showed that serum resistance in Haemophilus ducreyi type strain 35000HP required expression of the outer membrane protein DsrA because the isogenic dsrA mutant FX517 is highly serum susceptible. In this study, we confirmed this finding by construction of additional serum-susceptible dsrA mutants in more recently isolated serum-resistant strains. We also demonstrated that killing of dsrA mutants required an intact classical complement cascade but not the alternative or mannan-binding lectin pathways. Between 5- and 10-fold more purified human immunoglobulin M (IgM) but not IgG was deposited onto dsrA mutant FX517 than onto parent strain 35000HP, consistent with IgM initiation of the classical cascade. Depletion of IgM, but not IgG, from complement-intact serum inhibited killing of FX517. As predicted from the amounts of IgM bound, more of the individual complement components were bound by FX517 than by parent strain 35000HP. Examination of the binding of negative regulators of complement as an explanation for serum resistance indicated that parent strain 35000HP bound more C4 binding protein and vitronectin than FX517 but not factor H. However, the degree and pattern of complement component binding observed suggested that IgM binding to the serum-susceptible mutant FX517 was responsible for the activation of the classical pathway and the observed killing of FX517 as opposed to binding of negative regulators of complement by the serum-resistant parent. We speculate that an undefined neo-epitope, possibly carbohydrate, is exposed in the dsrA mutant that is recognized by naturally occurring bactericidal IgM antibodies present in human sera.
    Keywords: Haemophilus Ducreyi ; Haemophilus Ducreyi ; Haemophilus Ducreyi ; Immunoglobulin M ; Complement ; Immunoglobulin G ; Classical Pathway ; Vitronectin ; Outer Membrane Proteins ; Carbohydrates ; Complement Factor H ; Mannose-Binding Lectin ; Complement Activation ; Complement Component C4 ; Immunoglobulin M ; Complement ; Immunoglobulin G ; Classical Pathway ; Vitronectin ; Outer Membrane Proteins ; Carbohydrates ; Complement Factor H ; Mannose-Binding Lectin ; Complement Activation ; Complement Component C4 ; Immunoglobulin M ; Immunoglobulin G ; Classical Pathway ; Vitronectin ; Outer Membrane Proteins ; Carbohydrates ; Complement Factor H ; Mannose-Binding Lectin ; Complement Activation ; Complement Component C4 ; Immunogenetics ; Immune Response and Immune Mechanisms ; Bacteria ; Dsra Protein ; Dsra Protein;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 7
    In: Infection and Immunity, 2006, Vol. 74(4), p.2224
    Description: The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibody-dependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid.
    Keywords: Hemoglobin ; Hemoglobin Receptors ; Heme ; Chancroid ; Immunoglobulin G ; Animal Models ; Vaccines ; Iron ; Immunization ; Haemophilus Ducreyi ; Vaccination and Immunization ; Vaccines - Active Immunity ; Hgba Protein;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 8
    Language: English
    In: BioTechniques, 10/2000, Vol.29(4), pp.762-768
    Description: We present a simple and efficient RT-PCR method for the detection and quantitation of any poly(A)-containing mRNA that is not affected by contaminating genomic DNA and does not rely on exhaustive DNase digestion protocols. The technique described here requires the use of an antisense primer designed to contain 6-8 bp cDNA-specific sequence and an additional 17 Ts located on the 5' end to take advantage of the poly(A) tail. A second cDNA-specific sense primer can be used that does not need to be separated by intronic DNA sequence.
    Keywords: DNA, Complementary -- Genetics ; Poly A -- Analysis ; Polymerase Chain Reaction -- Methods ; RNA, Messenger -- Analysis;
    ISSN: 0736-6205
    E-ISSN: 1940-9818
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  • 9
    Language: English
    In: Methods in molecular biology (Clifton, N.J.), 2003, Vol.221, pp.161-8
    Keywords: DNA, Complementary -- Genetics ; Poly A -- Analysis ; RNA, Messenger -- Analysis ; Reverse Transcriptase Polymerase Chain Reaction -- Methods
    ISSN: 1064-3745
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 10
    Language: English
    In: Journal of Molecular Biology, 29 September 1994, Vol.242(4), pp.470-486
    Description: The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous protein which forms a collar/whiskers complex. Whiskers function as a helper protein for the long tail fibres assembly and plays a role in regulating retraction of the long tail fibres in response to environmental conditions. In this work we show that expression of the cloned wac gene in Escherichia coli yields a protein oligomer of 53 nm length which we call fibritin, and which is able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin indicates a 90% alpha -helical content, and scanning calorimetry shows that the protein has several distinct domains. The analysis of the 486 amino acid sequence of fibritin reveals three structural components: a 408 amino acid region that contains 12 putative coiled-coil segments with a canonical heptad (a-b-c-d-e-f-g) sub(n) substructure where the "a" and "d" positions are preferentially occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino acid residues, respectively) have no heptad substructure. The distribution of hydrophobic residues within heptads is more similar to a triple than to a double coiled-coil. The alpha -helical segments are separated by short "linker" regions, variable in length, that have a high proportion of glycine and proline residues. Each coiled-coil segment has, on the borders with linker regions, residues that are common to the N and C-terminal caps of the alpha -helices. Full-length and amino-terminally truncated fibritins can be reassembled in vitro after temperature-induced denaturation. Co-assembly of full-length fibritin and the N-terminal deletion mutant, as well as analytical centrifugation, indicates that the protein is a parallel triple-standard alpha -helical coiled-coil. Deletions of various N-terminal portions of fibritin did not block trimerisation but the mutant trimers are unable to bind to T4 particles. The last 18 C-terminal residues of fibritin are required for correct trimerisation of gpwac monomers in vivo. We propose that fibritin might serve as a convenient model for the investigation of folding and assembly mechanisms of alpha -fibrous proteins.
    Keywords: T4 Phage ; Fibritin ; Coiled-Coil Structure ; Sequence Analysis ; Protein Folding ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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