ABSTRACT
A monoclonal antibody obtained by immunization of mice with heat-killed cells of Listeria monocytogenes serotype 4d showed reactivity towards a protein (P45) from L. monocytogenes with an apparent molecular mass of 45 kDa. This protein was detected in the culture supernatant and at the cell surface of L. monocytogenes. Proteins cross-reacting with the monoclonal antibody were present in all Listeria strains investigated, except L. grayi. The structural gene was cloned in Escherichia coli and sequenced. Translation of the gene starts at a TTG initiation codon. The gene was found to code for a protein of 402 amino acid residues with a predicted molecular mass of 42.7 kDa. It has a signal peptide of 27 amino acid residues, resulting in a molecular mass for the mature polypeptide of 39.9 kDa. Protein database searches showed that this protein has 55% similarity and 38% identity to protein p60 of L. monocytogenes and exhibits significant sequence similarities to p54 from Enterococcus faecium and Usp45 from Lactococcus lactis. P45 was shown to have peptidoglycan lytic activity and the encoding gene was named spl (secreted protein with lytic property).
Subject(s)
Bacterial Proteins/analysis , Listeria monocytogenes/chemistry , N-Acetylmuramoyl-L-alanine Amidase/chemistry , Peptidoglycan/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cloning, Molecular , Genes, Bacterial , Immunoblotting , Mice , Molecular Sequence Data , Molecular WeightABSTRACT
Lipoteichoic acids (LTAs) of pathogenic and apathogenic Listeria species and of Staphylococcus aureus were fractionated and tested for their ability to stimulate production of cytokines (IL-1alpha, IL-6, TNF-alpha) in resident peritoneal macrophages (Mvarphi) of endotoxin-resistant C3H/HeJ mice using a serum-free medium. For IL-1alpha and IL-6 there were no detectable differences in the ability of LTA fractions of pathogenic and apathogenic Listeria species and of Staphylococcus aureus. However, LTA-2 fractions of Staphylococcus aureus, which might be less hydrophobic than the LTA-2 fractions of the listeriae-induced lower amounts of TNF-alpha. Furthermore, the more lipophilic LTA-2 fractions of all LTAs employed were more potent inducers of cytokines than the less lipophilic LTA-1 fractions. The biologic effect of LTAs appears, therefore, to depend mainly on their hydrophobicity.
ABSTRACT
A specific polyclonal antiserum was prepared against a gel-purified 60 kDa extracellular protein of Listeria monocytogenes ATCC 19111 corresponding to protein p60 previously detected in culture broths of L. monocytogenes strains Mackaness and EGD [Kuhn, M. & Goebel, W. (1989), Infection and Immunity 57, 55-61]. Indirect immunogold labelling combined with transmission electron microscopy and high-resolution scanning electron microscopy were used to investigate the location and distribution of p60 on the bacterial cell surface. In bacteria grown to the early stationary phase about 25% of the extracellular protein was estimated to be associated with the cell surface. The anti-p60 antiserum proved to be Listeria-specific. In an indirect immunofluorescence test the antiserum reacted with Listeria strains representing all species and different serotypes, except L. seeligeri, L. welshimeri, L. grayi and L. murrayi. No immunological cross-reactions were observed with 27 strains of bacteria from 16 other genera. The value of the anti-p60 antiserum in developing a diagnostic assay for Listeria cells in environmental samples and foods is discussed.
Subject(s)
Bacterial Proteins/metabolism , Listeria monocytogenes/metabolism , Antibodies, Bacterial , Antibody Specificity , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Cell Membrane/metabolism , Cross Reactions , Fluorescent Antibody Technique , Immunoblotting , Listeria monocytogenes/immunology , Listeria monocytogenes/ultrastructure , Microscopy, Electron, Scanning , Microscopy, Immunoelectron , Molecular WeightABSTRACT
Lipoteichoic acids were isolated from eleven species of the genus Staphylococcus using phenol-water partition and hydrophobic chromatography on octyl-Sepharose CL-4B. The lipoteichoic acids purified could be visualized by SDS-PAGE. They were shown to be composed of a hydrophilic poly(glycerophosphate) chain covalently linked to gentiobiosyldiacylglycerol, the common lipid anchor of these molecules. Glycerophosphate units of the hydrophilic chain were found to be partly substituted with ester-linked D-alanine, except in the case of S. cohnii. The lipoteichoic acids isolated from S. cohnii, S. hominis, S. saprophyticus and S. simulans contain alpha(1-2)-linked N-acetylglucosamine as an additional substituent of the poly(glycerophosphate) backbone.
Subject(s)
Lipopolysaccharides/chemistry , Staphylococcus/analysis , Teichoic Acids/chemistry , Chromatography, Gas , Chromatography, Gel , Chromatography, Thin Layer , Electrophoresis, Polyacrylamide Gel , Lipopolysaccharides/isolation & purification , Molecular Structure , Teichoic Acids/isolation & purificationABSTRACT
PAH secretion (TPAH) was studied in rats at spontaneously occurring glomerular filtration rate (GFR). At saturated transport, TPAH was found to be correlated to GFR. This relationship was also observed at unsaturated transport where TPAH depends upon the PAH concentration in arterial plasma. However, no significant correlation between TPAH and renal PAH load or renal plasma flow rate was found when the effects of GFR were removed by partial correlation analysis. A dependency of TPAH on GFR explains the correlations found between filtration fraction (FF) and renal PAH extraction (EPAH) or renal tubular PAH extraction fraction (EPAH--FFPAH). Thus, even at low PAH concentration in a. plasma, renal PAH extraction may only be assumed to be constant if the filtration fraction is constant.
Subject(s)
Aminohippuric Acids/metabolism , Glomerular Filtration Rate , Kidney/metabolism , p-Aminohippuric Acid/metabolism , Animals , Biological Transport , Kidney Tubules/metabolism , Male , Osmolar Concentration , RatsABSTRACT
Inactin, given to rats for anaesthesia at a dose of 100 mg per kg body weight results in an arterial plasma concentration of 10.6 +/- 1.14 mg%, measured 40 min after the onset of anaesthesia. When similar concentrations of Inactin or Amytal were applied to frog skins the decrease in the in vitro short circuit current is dose dependent. With Inactin, the inhibition was, at least in part, reversible and the electrical conductance was not affected. Amytal however was found to depress SCC and electrical conductance irreversibly. The data are discussed in regard to active renal transport processes.
Subject(s)
Barbiturates/blood , Membrane Potentials/drug effects , Skin Physiological Phenomena , Amobarbital/blood , Amobarbital/pharmacology , Animals , Barbiturates/pharmacology , Electric Conductivity/drug effects , Kidney Tubules/physiology , Male , Rana esculenta , RatsABSTRACT
Net secretion rate of para-aminohippurate (PAH) in the proximal convolution of the rat kidney changes concomitantly with single nephron glomerular filtration rate (GFR) and intratubular flow rate. Reabsorption of PAH in the proximal convolution is negligible. The PAH concentration profile along the length of the proximal convolution does not change markedly with variations in GFR. Net PAH secretion by single nephrons, measured at the end of proximal convolutions, is about one-half that measured at the beginning of distal convolutions and in final urine. As in the entire kidney, at constant renal plasma flow and concentration of PAH, renal secretion rate of PAH also changes concomitantly with GFR. It is concluded that PAH secretion along the loop of Henle (i.e., probably along the pars recta) is also related to single nephron GFR, as is PAH secretion in the proximal convolution.
