Association of a Bacteriophage with Meningococcal Disease in Young Adults

Despite being the agent of life-threatening meningitis, Neisseria meningitidis is usually carried asymptomatically in the nasopharynx of humans and only occasionally causes disease. The genetic bases for virulence have not been entirely elucidated and the search for new virulence factors in this species is hampered by the lack of an animal model representative of the human disease. As an alternative strategy we employ a molecular epidemiological approach to establish a statistical association of a candidate virulence gene with disease in the human population. We examine the distribution of a previously-identified genetic element, a temperate bacteriophage, in 1288 meningococci isolated from cases of disease and asymptomatic carriage. The phage was over-represented in disease isolates from young adults indicating that it may contribute to invasive disease in this age group. Further statistical analysis indicated that between 20% and 45% of the pathogenic potential of the five most common disease-causing meningococcal groups was linked to the presence of the phage. In the absence of an animal model of human disease, this molecular epidemiological approach permitted the estimation of the influence of the candidate virulence factor. Such an approach is particularly valuable in the investigation of exclusively human diseases.     

CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.     

Ambrosiozyma kamigamensis sp. nov. and A. neoplatypodis sp. nov., two new ascomycetous yeasts from ambrosia beetle galleries

Two new yeast species of the genus Ambrosiozyma are described on the basis of comparison of nucleotide sequences of large subunit of ribosomal DNA D1/D2 region. Ambrosiozyma kamigamensis and Ambrosiozyma neoplatypodis differ from Ambrosiozyma ambrosiae by 17 nucleotides (3.0%) and 16 nucleotides (2.8%), respectively, out of 565. The two species differ from each other by 13 nucleotides. Ambrosiozyma kamigamensis was isolated from galleries of the ambrosia beetle, Platypus quercivorus, in specimens of Quercus laurifolia and Castanopsis cuspidata located in the southern part of Kyoto, Japan. Ambrosiozyma neoplatypodis was isolated from similar material, but only in Q. laurifolia. Ambrosiozyma kamigamensis can be distinguished from the other Ambrosiozyma species by the inability to assimilate erythritol, whereas A. neoplatypodis can be distinguished by the ability to assimilate both L: -arabinose and nitrate. The type strains of A. kamigamensis and A. neoplatypodis are JCM 14990(T) (=CBS 10899(T)) and JCM 14992(T) (=CBS 10900(T)), respectively. This is the first report of new Ambrosiozyma species since the genus was proposed.     

An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic alpha-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.     

Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30–40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people. Received: 7 February 2001 / Accepted: 3 April 2001     

Characterization of cellulases in an extracellular fraction from Trichoderma reesei under conditions of isoelectric focusing (IEF)

Abstract A cellulase-containing fraction present in the culture fluid of Trichoderma reesei grown on cellulose was obtained by fractionated centrifugation. The buoyant density of this fraction was D = 1.060 g/ml. Its ultrastructural properties, as detected by transmission electron microscopy, are given. The fraction consists of membrane vesicles attached to a carbohydrate polymer. This polymer is positive to Ruthenium red staining.
The effect of urea on the extraction and separation of acidic cellulases from this fraction is described. Linear gradient gels for both urea (up to 8.0 M urea) and polyacrylamide gels (up to 30%) were used to determine adequate separation conditions for isoelectric focusing (IEF) in a polyacrylamide gel matrix. The effect of urea on the extraction and separation conditions was tested by titration curves. In the presence of 6.0−8.0 M urea, the main cellulase-containing hydrolase complex (pI_app4.2) from this fraction is split into 3 isoenzymes and a further cellulase (pI 5.65).     

Experimentelle Untersuchungen über die Wirkung innersekretorischer Organe

Ohne Zusammenfassung