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  • 1
    Digitale Medien
    Digitale Medien
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 135-148 
    ISSN: 0173-0835
    Schlagwort(e): Two-dimensional polyacrylamide gel electrophoresis ; Haemophilus influenzae ; Bacteria ; Protein database ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Chemie und Pharmazie
    Notizen: The proteins of nontypable and type b Haemophilus influenzae isolates were characterised using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Coomassie Brilliant Blue R-250 was used for protein detection. Two hundred and twenty eight proteins were resolved from whole cell lysates prepared from a standard nontypable H. influenzae strain (designated HI-64443) when isoelectric focusing was used for the first-dimensional separation of 2-D PAGE. When nonequilibrium pH gel electrophoresis (NEPHGE) was used to separate basic proteins in the first dimension, 50 proteins were detected for HI-64443; 20 of the basic proteins detected were considered to be unique for this separation protocol. The apparent molecular weights and isoelectric points were determined for 82 of the proteins resolved for HI-64443. The variation of the proteins from the standard bacterial strain (HI-64443) was determined for nontypable H. influenzae isolates. On the basis of their electrophoretic mobilities, 17.5% of the proteins of HI-64443 were shared by four other non-typable H. influenzae strains analysed. These data identified both conserved and variable proteins among the nontypable H. influenzae isolates analysed. The results obtained indicated that 2-D PAGE was able to discriminate nontypable H. influenzae into population clones identified by other procedures. The 2-D protein profiles obtained for type b H. influenzae strains were similar to those obtained for nontypable H. influenzae strains. The extent of the protein variation observed between type b and nontypable H. influenzae strain was similar to that observed among nontypable strains alone. These data are discussed in relation to the application of 2-D PAGE as a tool for studies on bacterial epidemiology and for the analysis of the genome structure and gene expression of Haemophilus influenzae.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
    ISSN: 0173-0835
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Chemie und Pharmazie
    Notizen: Serotype 5 coxsackie B virus (CBV5) can establish in vitro persistent infections in human rhabdomyosarcoma (RD) cells. This paper describes the characterisation of the virus released from the persistently infected RD cell line designated piRD-3673. Although infectious virus was released for 42 sequential passages of piRD-3673 cells, no gross virus-specific cytopathic effect was detected when the cells were examined by light microscopy. Two-dimensional polyacrylamide gel electro-phoresis was used to compare the virus released from piRD-3673 cells with the CBV5 isolate (CBV-3673) used to initiate the persistent virus infection. Two of the virus intracellular proteins (apparent molecular weights 33 000 and 39 000, designated p33 and p39, respectively) increased in their net basic charge for the virus released from piRD-3673 cells compared to CBV-3673; a reduction in the apparent molecular weights of p33 and p39 was also observed. The charge alteration for both p33 and p39 was a two-stage process, the accumulative effect of which resulted in p33 increasing in pI from 6.14 to 6.53 and p39 increasing in pI from 6.29 to 6.63. The first mutation of p33 and p39 occurred between passages 7 and 10 of piRD-3673 cells and affected both the charge and apparent molecular weight of these two proteins. The second mutation at passage 15 of piRD-3673 cells caused only a change in the charge of p33 and p39. Two other virus proteins (p54 and p75) showed no evidence of mutation over the same passage history of piRD-3673 cells. The virus released from piRD-3673 cells also differed from CBV-3673 by two further criteria, a reduction in plaque-forming efficiency in HEp-2 cells and increased virus replication in RD cells. These data on virus evolution are discussed in relation to the maintenance of persistent CBV infections and the presence of naturally occurring CBV variants.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 3
    ISSN: 0173-0835
    Schlagwort(e): Two-dimensional polyacrylamide gel electrophoresis ; Coxsackie B virus ; Protein synthesis ; Persistent virus infection ; T cells ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Chemie und Pharmazie
    Notizen: Coxsackie B viruses (CBV) have been implicated in various human diseases that present as either limited acute infections or prolonged chronic infections. A number of investigations have suggested that a virus-induced immune dysfunction might play a role in in vivo pathogenesis. In the current study, we describe CBV infection of two human T cell-derived cell lines (Jurkat and MOLT-4 cells) as potential models for CBV infection of lymphocytes. Short term (up to 144 h post-infection) CBV infection of either cell line resulted in a decline in the viability of the cell population together with an approximate 10-fold rise in the titre of infectious virus during the period of incubation. Analyses of the intracellular proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) demonstrated that although putative virus proteins were detectable there was minimal inhibition of the cellular protein synthesis following CBV infection. This contrasted with the more permissive and highly lytic CBV infection of HEp-2C cells (Cash, Electrophoresis 1991, 10, 793-800). Persistently infected cell lines from both Jurkat and MOLT-4 cells (piJURKAT-3673 and piMOLT-2667 cells) were established. Analyses of intracellular protein synthesis of these persistently infected cell lines showed the synthesis of novel proteins not detected for the corresponding uninfected parental cell line. There were no significant alterations in overall cellular protein synthesis detectable by the small format 2-D PAGE system employed in these investigations. The data presented in the current investigation will contribute towards studies on virus-induced responses of specific biological functions associated with T cells.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 4
    Digitale Medien
    Digitale Medien
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1472-1482 
    ISSN: 0173-0835
    Schlagwort(e): Haemophilus influenzae ; Proteins ; 2-D PAGE ; Mutant ; Superoxide dismutase ; Proteome ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Chemie und Pharmazie
    Notizen: Members of the Haemophilus genus are responsible for various human infections including respiratory infections and meningitis. The complete nucleotide sequence of the Rd strain of Haemophilus influenzae has been reported and represents a valuable resource to investigate gene expression within this bacterial group. We described previously the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to characterise the proteins of Haemophilus influenzae (Cash et al., Electrophoresis 1995, 16, 135-148). We have extended these data with comparative studies of the proteins from other members of the Haemophilus genus (specifically H. parainfluenzae, H. haemolyticus and H. parahaemolyticus) to identify homologous proteins and, by extension, the genes encoding them, among these bacteria. The proteins extracted from each of these bacterial isolates were compared by coelectrophoresis to the 2-D protein profile of the reference nontypable strain of H. influenzae (HI-64443) used as the basis for the 2-D protein database. A composite reference 2-D protein profile of HI-64443 was derived from three independent analyses of the soluble bacterial proteins. Between 21% and 37% of the HI-64443 proteins from the reference 2-D protein profile comigrated with proteins in the other isolates from the Haemophilus genus. This compared with 62% and 64% comigration when HI-64443 was compared with the Eagan and Rd strains of H. influenzae, respectively. The 2-D protein profile of the Rd strains of H. influenzae was compared to that of HI-64443 by coelectrophoresis; 64% of the proteins detected for the Rd strain comigrated with proteins found for HI-64443 when analysed in parallel. The capacity of 2-D PAGE to investigate global interactions of gene expression was applied to the analysis of superoxide dismutase (SOD) expression in H. influenzae strain Eagan. A “knock-out” mutant in the sodA gene which encodes [Mn]-SOD was characterised with respect to proteins synthesis compared to the parental isolate. From these analyses, the primary product of sodA was provisionally identified as a protein with a molecular mass of 25 500 Da and an estimated pI of 6.55. Quantitative changes in the expression of two other proteins in the SOD mutant were detected by comparison with the parental isolate. These data are discussed in relation to the development of a 2-D protein database for H. influenzae and related bacteria to investigate genome homologies and gene expression.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 5
    Publikationsdatum: 2011-10-01
    Print ISSN: 1862-8346
    Digitale ISSN: 1862-8354
    Thema: Biologie , Medizin
    Publiziert von Wiley
    Standort Signatur Erwartet Verfügbarkeit
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