Secretion of periplasmic PhoA protein during its supersynthesis into the medium using outer membrane vesicles. Features of chemical composition of the vesicles and membranes of Escherichia coli secreting cells

E. coli K12802 cells transformed by multicopy plasmid with phoA gene acquire the ability to oversynthesize alkaline phosphatase, secrete it into the cultural medium, and accumulate the precursor of this enzyme. The dynamics of enzyme production and secretion as well as cytomorphological changes revealed the existence of a mechanism of selective enzyme secretion into the medium. It is characterized by a decrease of enzyme specific activity in periplasm and its increase in cultural medium, appearance of numerous local zones of adhesion of cytoplasmic and outer membranes, formation of large extracellular outer membrane vesicles containing PhoA protein on the cell poles, and their release into the medium. We isolated the vesicles and found that they contain PhoA (in dominating quantity), several other periplasmic proteins, and matrix proteins of outer membranes. By their phospholipid and protein composition, they correspond to the fraction of outer membranes which have the largest density and sedimentation rate and, apparently, contain no lipoprotein.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

Isolation and Characterization of a New Extracellular Bacteriolytic Endopeptidase of <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL1

The previously unstudied bacteriolytic enzyme L(4) was isolated from the culture liquid of the bacterium Lysobacter sp. XL1 in electrophoretically homogeneous state. The enzyme L(4) is a diaminopimelinoyl-alanine endopeptidase relative to peptidoglycan of Lysobacter sp. XL1. The enzyme is an alkaline protein of approximately 21 kD. The N-terminal amino acid sequence of the enzyme has been determined - A V V N G V N Y V Gx T T A ... The maximal activity of the enzyme was observed in 0.05 M Tris-HCl at pH 8.0 and 50-55 degrees C. The half-inactivation temperature of the enzyme is 52 degrees C. The endopeptidase L(4) is not a metalloenzyme since it is not affected by EDTA. The enzyme is inhibited by p-chloromercuribenzoic acid by 72% and by phenylmethylsulfonyl fluoride by 43%, which indicates the involvement of serine and thiol groups in its functioning.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

The Effect of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Negative Bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells was altered by treating them with polymyxin B.     

Extracellular yeast-lytic enzyme of the bacterium <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL 1

An enzyme exhibiting yeast-lytic activity has been isolated from the culture liquid of the bacterium Lysobacter sp. XL 1. The optimal conditions for the hydrolysis of Saccharomyces cerevisiae cells by the enzyme have been established: 0.15 M sodium acetate buffer, pH 6.0, 50 degrees C. The yeast-lytic activity of the enzyme is inhibited by EDTA, p-chloromercuribenzoate, and phenylmethylsulfonyl fluoride. According to the data of SDS-PAGE, the molecular weight of the protein is 36 kD. The enzyme hydrolyzes casein, hemoglobin, and synthetic peptide Abz-Ala-Ala-Phe-pNA, i.e. it exhibits proteolytic activity. The properties of the enzyme and its molecular weight correspond to those of a previously isolated extracellular metalloproteinase. The N-terminal amino acid sequence of the protein exhibits 67% homology with the N-terminal sequence of achromolysine of Achromobacter lyticus (EC 3.4.24.-).     

Transformation of various strains of Escherichia coli by multicopy plasmids with the phoA gene leads to secretion of a periplasmatic alkaline phosphatase into the media and accumulation of its precursor

Various E. coli strains have been transformed by multicopy plasmids pHI-1, pHI-7 and pPHO I carrying the entire regulatory and structural phoA sequences. All the transformants with the intact pho regulatory system have been shown to be capable of alkaline phosphatase oversynthesis and secretion into the medium. They are also able to accumulate the alkaline phosphatase precursor localized in the outer membrane fraction. The transformants of the restriction or recombination mutants are the most efficient producers of the extracellular enzyme and its precursor.     

RAPID SCREENING FOR SALMONELLA IN FISHMEAL BY ENRICHMENT-IMMUNOASSAY

We previously described an enrichment-immunoassay utilizing a T6 monoclonal antibody capture enzyme-linked immunosorbent assay. Here we evaluated it for the rapid screening for Salmonella in fishmeal obtained from the national Animal and Plant Quarantine service in the People's Republic of China. In this method, the number of Salmonella present is first expanded by appropriate enrichment cultures, and the pathogens are then directly detected by the T6 immunoassay. In a total of 94 enrichment cultures of fishmeal, we obtained an overall concordance of 98% between the results obtained in parallel by this method and by conventional test method. The positive prediction by this method was 92% and the negative prediction was 100%. The turn around time for the new test was 27 h which is a significant improvement from the turn around time exceeding 96 h required for the conventional test method. This test proved to be compatible with the routine work flow in the practical setting of a quarantine laboratory.     

Mannose binding lectin plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells

Background  

Mannose binding lectin (MBL) is an important host defence protein against opportunistic fungal pathogens. This carbohydrate-binding protein, an opsonin and lectin pathway activator, binds through multiple lectin domains to the repeating sugar arrays displayed on the surface of a wide range of clinically relevant microbial species. We investigated the contribution of MBL to antifungal innate immunity towards C. parapsilosis in vitro.