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Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis. Geneviève Renauld-Mongénie, 2004.A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6 . Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf) . These two domains are conserved in an alignment of different TbpB sequences from N . meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains . Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding . Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements . The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains . Efficacy of Novel Hemagglutinin-Neuraminidase Inhibitors BCX 2798 and BCX 2855 against Human Parainfluenza Viruses In Vitro and In Vivo. Irina V. Alymova, 2004.Human parainfluenza viruses are important respiratory tract pathogens, especially of children . However, no vaccines or specific therapies for infections caused by these viruses are currently available . In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein . The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK2 cells . The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 µM in HA inhibition assays and from 0.02 to 20 µM in NA inhibition assays . The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 µM . BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA . In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death . Treatment beginning 24 h after the start of infection did not prevent death . Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans . High-Performance Liquid Chromatography Analyses of Pyoverdin Siderophores Differentiate among Phytopathogenic Fluorescent Pseudomonas Species. Alain Bultreys, 2003.The relationship of pyoverdins produced by 41 pathovars of Pseudomonas syringae and by phytopathogenic Pseudomonas species was investigated . A high-performance liquid chromatography method for analyzing the culture medium proved to be superior to isoelectric focusing for detecting pyoverdin production, for differentiating slightly different pyoverdins, and for differentiating atypical from typical Fe(III)-chelated pyoverdins . Nonfluorescent strains were found in Pseudomonas amygdali, Pseudomonas meliae, Pseudomonas fuscovaginae, and P . syringae . Pseudomonas agarici and Pseudomonas marginalis produced typical pyoverdins . Among the arginine dihydrolase-negative fluorescent Pseudomonas species, spectral, amino acid, and mass spectrometry analyses underscored for the first time the clear similarities among the pyoverdins produced by related species . Within this group, the oxidase-negative species Pseudomonas viridiflava and Pseudomonas ficuserectae and the pathovars of P . syringae produced the same atypical pyoverdin, whereas the oxidase-positive species Pseudomonas cichorii produced a similar atypical pyoverdin that contained a glycine instead of a serine . The more distantly related species Pseudomonas asplenii and Pseudomonas fuscovaginae both produced a less similar atypical pyoverdin . The spectral characteristics of Fe(III)-chelated atypical pyoverdins at pH 7.0 were related to the presence of two ß-hydroxyaspartic acids as iron ligands, whereas in typical pyoverdins one of the ligands is always ornithine based . The peptide chain influenced the chelation of iron more in atypical pyoverdins . Our results demonstrated that there is relative pyoverdin conservation in the amino acids involved in iron chelation and that there is faster evolution of the other amino acids, highlighting the usefulness of pyoverdins in systematics and in identification . Differential Transfer and Dissemination of Hypovirus and Nuclear and Mitochondrial Genomes of a Hypovirus-Infected Cryphonectria parasitica Strain after Introduction into a Natural Population. Patrik J. Hoegger, 2003.Biological control of plant diseases generally requires release of living organisms into the environment . Cryphonectria hypoviruses function as biological control agents for the chestnut blight fungus, Cryphonectria parasitica, and hypovirus-infected C . parasitica strains can be used to treat infected trees . We used naturally occurring molecular marker polymorphisms to examine the persistence and dissemination of the three genomes of a hypovirus-infected C . parasitica strain, namely, the double-stranded RNA genome of Cryphonectria hypovirus 1 (CHV1) and the nuclear and mitochondrial genomes of its fungal host . The hypovirus-infected strain was experimentally introduced into a blight-infested chestnut coppice forest by treating 73 of 246 chestnut blight cankers . Two years after introduction, the hypovirus had disseminated to 36% of the untreated cankers and to 35% of the newly established cankers . Spread of the hypovirus was more frequent within treated sprout clusters than between sprout clusters . Mitochondrial DNA of the introduced fungus also was transferred into the resident C . parasitica population . Concomitant transfer of both the introduced hypovirus and mitochondrial DNA was detected in almost one-half of the treated cankers analyzed . The introduced mitochondrial DNA haplotype also was found in three resident isolates from newly established cankers . The nuclear genome of the introduced strain persisted in the treated cankers but did not spread beyond them . Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction. Hebe M. Dionisi, 2003.The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification . DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter . Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix . The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2% . Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay . Instead, a larger variability was associated with the PCR assay . The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors . Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes .
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