ABSTRACT
Phages are excellent models for studying the mechanism of DNA replication in prokaryotes. Identification of phage proteins involved in phage DNA replication is the first prerequisite for elucidation of the phage replication module. We focused on replication proteins gp41 (a putative helicase from SF2 superfamily), gp43 (a RepA-like protein), and gp44 (a putative DNA polymerase A) of phage BFK20 grown in Brevibacterium flavum. To identify them in the phage-host system, we prepared antibodies to these proteins which were cloned and expressed in Escherichia coli as his-tagged recombinant proteins. After purification to homogeneity the recombinant proteins served for raising specific polyclonal antibodies in mice. Using these antibodies in Western blot analysis the phage proteins gp41, gp43 and gp44 were detected during the phage growth cycle. The proteins gp41 and gp43, prepared from cell lysate by ammonium sulphate precipitation, were N-terminally sequenced and found to contain the sequences N-SVKPRELR-C and N-MLGSTML-C, respectively. This means that gp41 starts with serine but not with common methionine. We consider these findings an initial but important step towards more thorough characterization of replication proteins of phage BFK20.
Subject(s)
Bacteriophages/genetics , Brevibacterium flavum/virology , Siphoviridae/genetics , Viral Proteins/analysis , Animals , Antibodies, Viral/analysis , Antibodies, Viral/immunology , Bacteriophages/physiology , Immunization , Immunoblotting , Mice , Mice, Inbred C57BL , Viral Proteins/genetics , Viral Proteins/immunology , Virus ReplicationABSTRACT
Polysaccharide and lipid A are responsible for the wide-ranging pharmacological activity of bacterial lipopolysaccharides (LPS). The alterations in LPS structure result in various effects on different functions of the target cells. The effects of LPS substructures, the polysaccharide (P) and lipid A (L) from E. coli on the innate mechanisms of human leucocytes were examined and compared in this study. Incubation of leucocytes with LPS and L and P analogues (1 and 100 microg/ml) enhanced their biological activity in dependence on their structure. These results showed that LPS was a less active immunomodulator of leucocytes than L and P analogues isolated from E. coli strains adapted to antimicrobial agents.
Subject(s)
Escherichia coli/chemistry , Immunologic Factors/pharmacology , Leukocytes/drug effects , Lipid A/pharmacology , Lipopolysaccharides/pharmacology , Blood Bactericidal Activity , Colony Count, Microbial , Humans , Immunologic Factors/chemistry , In Vitro Techniques , Leukocytes/enzymology , Leukocytes/metabolism , Lipid A/chemistry , Lipopolysaccharides/chemistry , Muramidase/metabolism , Phagocytosis/drug effectsABSTRACT
Resistant strains of Escherichia coli were obtained by stepwise cultivation in media with increasing concentration of antimicrobially active 1-(methyldodecyl)dimethylamine oxide and 1-(methyldodecyl)trimethylammonium bromide. Adaptive changes were determined in the fatty-acid (FA) composition in an isolated lipopolysaccharide sample from the outer membrane of these strains. The composition of this FA mixture from adapted strains was compared with that of FA from a sensitive strain. The differences were found in level of palmitic, heptadecanoic, heptadecenoic, heptadecadienoic and nonadecenoic acids. In addition, the adapted strains differed from each other in the content of myristic, pentadecanoic, stearic and linoleic acids.
Subject(s)
Dimethylamines/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Fatty Acids/metabolism , Quaternary Ammonium Compounds/pharmacology , Adaptation, Physiological/drug effects , Drug Resistance, Bacterial , Lipopolysaccharides/metabolismABSTRACT
The study presents comparison of immunomodulatory effects of Staphylococcus aureus, Escherichia coli and Candida albicans disintegrated cells on selected immune mechanisms of human and mouse leukocytes. We measured their phagocytic activity, phagocytic index and microbicidal activity against Staphylococcus aureus, Escherichia coli and Candida albicans cells as well as peroxidase and lysozyme activities of human and mouse leukocytes. Our results revealed predominantly inhibitory effect of disintegrated microorganisms on nonspecific immune functions of human leukocytes, but mainly stimulatory effect on mouse leukocytes monitored immune functions.
Subject(s)
Adjuvants, Immunologic , Candida albicans/immunology , Escherichia coli/immunology , Immunity, Cellular , Staphylococcus aureus/immunology , Animals , Blood Bactericidal Activity , Humans , Leukocytes/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , PhagocytosisABSTRACT
Crude extracts obtained from the stem bark of Mahonia aquifolium have been investigated as to the chemical composition and anticomplementary activity. The results show that their anticomplementary activity is mainly due to the alkaloid components. Especially the BBI alkaloid fraction and berberine showed a strong inhibitory effect on CH50 total hemolytic complement assay. The crude extract of M. aquifolium was less active than berberine or the fraction BBI alkaloids. The results indicate that the fraction of BBI alkaloids and berberine largely account for the immunomodulatory activity of the crude extract of M. aquifolium.
