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Biomedical subjects

M Thelestam

Publications and source records attributed to M Thelestam.

89 records · Page 5Linked to original sources
PubMed1980

Effect of tobacco smoke compounds on the plasma membrane of cultured human lung fibroblasts.

The ability of compounds derived from tobacco and tobacco smoke to increase the permeability of the membranes of human lung fibroblasts has been studied by measuring the release of an intracellular marker after short term exposure. Of the 464 compounds tested, about 25% gave rise to severe membrane damage. The most active compounds, when divided according to functionality, were found within the groups of amines, strong acids and alkylated phenols, whereas nitriles and polycyclic aromatic hydrocarbons were found completely inactive. A pronounced effect of the chain length on the activity was observed for the aliphatic alcohols, aldehydes and acids, and all monocyclic aromatic compounds but benzonitriles and benzoic acids showed an increase in activity with increasing alkylsubstitution. It is concluded that tobacco smoke contains a number of membrane damaging substances. These membrane active compounds could not only cause direct toxic reactions but also potentiate the toxic effect by promoting the cell membrane penetration of other toxic substances in tobacco smoke.

Cell Membrane↗
PubMed1980

Screening and characterization of membrane damaging effects in tissue culture.

A leakage test system for specific detection, measurement and characterization of membrane damage in human lung fibroblasts is discussed. Examples of applications with various types of substances are presented, i.e. tobacco smoke compounds; animal, plant and microbial cytolysins; membrane damaging antibiotics.

Anti-Bacterial Agents↗
PubMed1980

Interaction of streptolysin O from Streptococcus pyogenes and theta-toxin from Clostridium perfringens with human fibroblasts.

The membrane-damaging properties on human diploid embryonic lung fibroblasts of streptolysin O (from Streptococcus pyogenes) and theta-toxin (from Clostridium perfringens) were compared. The results are consistent with the suggested mechanism for hemolysis by streptolysin O involving one fixation site and one lytic site of this cytolysin. However, the membrane-damaging activity of the two toxins differed with respect to (i) relative cytolytic activity on human diploid lung fibroblasts compared with that on sheep erythrocytes, (ii) binding to the fibroblast membrane, (iii) activity at 0 degrees C, (iv) membrane repair after more than 30 min, and (v) effect on influx of amino acids. It is concluded that the mechanism of membrane damage caused by theta-toxin differs from that of cytoplasmic membrane. These results question the current concept that all thiol-activated, cholesterol-inactivated bacterial toxins are similar both structurally and functionally.

Binding Sites↗
PubMed1980

Interaction of cytopathogenic toxin from Clostridium difficile with cells in tissue culture.

Partially purified cytopathogenic toxin from Clostridium difficile induced morphological changes in five cell lines in tissue culture. The relative sensitivity scale of the cell lines was human lung and intestinal fibroblasts greater than Chinese hamster ovary cells much greater than mouse adrenal cells greater than mouse neuroblastoma cells. The cytopathogenic effect did not occur in toxin-treated lung fibroblasts incubated at 0 degree C. Pre-incubation of lung fibroblasts with 2,4-dinitrophenol prevented the cytopathogenic effect. The toxin bound to as yet unidentified receptors at the surface of human lung and intestinal fibroblasts. The toxin-induced morphological (actinomorphic) changes in lung and intestinal fibroblasts closely resembled the effects induced by the fungal metabolite cytochalasin B (CB), which is known to disrupt microfilaments reversibly. Indirect immunofluorescence with anti-actin antiserum demonstrated that the C. difficile toxin disrupted the straight actin filament bundles seen in normal fibroblasts. The cytopathogenic effect became apparent 3--5 h after exposure to toxin. However, irreversible intoxication occurred already within 20 min of exposure, as toxin-treated fibroblasts which were trypsinized and reseeded were not able to attach to the solid substratum and regenerate their typical shape, a process requiring reorganization of actin into microfilament bundles. Two possible different modes of action of the toxin, leading to microfilament disruption, are suggested: 1) Transmembrane signal by surface-bound toxin via microfilament-linked integral membrane protein(s) and 2) Penetration of surface-bound whole toxin or an active fragment, followed by its intracellular action. The experimental evidence so far is consistent with either of these mechanisms.

Animals↗
PubMed1979

Classification of microbial, plant and animal cytolysins based on their membrane-damaging effects of human fibroblasts.

38 cytolytic agents of mainly microbial origin were investigated with respect to membrane-damaging activity on human diploid fibroblasts. Increased plasma membrane permeability was measured as leakage of three defined cytoplasmic markers of various sizes: alpha-aminoisobutyric acid, uridine nucleotides and ribosomal RNA. The relative leakages of these markers, caused by different concentrations of the various cytolysins, yielded a leakage pattern for each substance. Five distinct types of leakage patterns were obtained. These were transformed into numerical expressions by calculating the ratios between the amounts of cytolysin needed to release 50% of the nucleotide and ribosomal RNA markers and the amounts required to release 50% of the alpha-aminoisobutyric acid marker (ED50 ratios). A classification of the cytolysins into five groups was arrived at on the basis of the different types of leakage patterns with the aid of reference cytolysins with well-known mechanisms of membrane interaction. These groups comprised: (1) detergent-like agents, (2) agents interacting with only certain constituents of the cell membrane, (3) agents interacting with specific receptor molecules in the membrane, (4) agents inducing small functional holes of a definable size, and (5) agents inducing only a very limited increase in plasma membrane permeability. The system may be useful for characterization and differentiation of new cytolytic agents of various sources as it divides membrane-damaging agents into separate groups on the basis of their principal function on intact human cells.

Animals↗
PubMed1977

Cytopathogenic effects of atoxyl, an ototoxic compound, on human diploid fibroblasts in vitro.

Atoxyl, an arsenic compound, may cause degeneration in vivo of the inner ear including cells of the stria vascularis and hair cells. The mechanism behind the cytotoxic effect is not known. The effects of atoxyl at the subcellular level were investigated in this study using human diploid embryonic lung fibroblasts in monolayer cultures as an in vitro model system. Atoxyl caused a subtle but significant increase in the permeability of the fibroblast plasma membrane, as measured by release of a low molecular weight cytoplasmic marker (alpha-amino isobutyric acid). At higher concentrations or after longer incubation times, protein synthesis was impaired. This effect occurred in parallel with alterations in the cellular morphology as viewed by light microscopy. In the final stages of atoxyl intoxication the cells released also a higher molecular weight marker (nucleotide), indicating a further increased membrane permeability following the primary damage. It is concluded that atoxyl exerts a dual effect on the human fibroblasts, namely on membrane permeability and protein synthesis. Although the concentrations used were higher than those exerting the ototoxic effects in vivo, the prolonged exposure times to low concentrations obtained in whole animals may very well compensate for this fact. The effects observed in the in vitro fibroblast model system may thus be relevant to the mechanism of action of atoxyl during induction of ototoxic effects in vivo.

Aniline Compounds↗
PubMed1976

Cytotoxic effects on the plasma membrane of human diploid fibroblasts--a comparative study of leakage tests.

Confluent monolayers of human diploid lung fibroblasts were treated with cytolytic agents. The induced membrane damage was investigated by different test systems. Changes of membrane permeability were compared with morphological alterations. Four tests employed leakage of cytoplasmic markers of different sizes as criteria of membrane damage. Radioactive markers of the following decreasing size order were used: [3H]RNA (MW greater than 200,000) greater than 51Cr greater than [3H]nucleotides greater than [1-14C]alpha-amino-isobutyric acid (AIB, MW 103). Uptake of trypan blue was employed as a fifth criterior of changed membrane permeability. A comparison between the tests indicated the following order of sensitivity for detection of membrane damage: leakage of AIB-label greater than leakage of nucleotide label greater than leakage of 51Cr-label=uptake of trypan blue= morphological changes greater than leakage of RNA-label. Two distinct types of leakage patterns were evident: 1. Upon incubation with Triton X-100 all four release curves coincided. This was considered as representing large functional "holes". 2. With the polyene amphotericin B the smallest marker (AIB) was released to a strikingly greater extent than other markers. This was regarded as representing very small functional "holes". Melittin from bee venom and theta-toxin from Clostridium perfringens induced leakage patterns of two intermediate types. The results indicate that a combination of several different size markers may be useful for characterizing induced membrane lesions.

Amphotericin B↗
PubMed1975

Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts.

Human diploid lung fibroblasts were treated with cytolytic bacterial toxins and the nature of the membrane damage was investigated. [3H] uridine was used for differential labeling of cytoplasmic components of small or large molecular size. Two principal size categories were achieved by labeling the fibroblasts in either early growth phase or stationary phase, a high-molecular weight ribonucleic acid label and a low-molecular-weight nucleotide label. The size of the labeled molecules was determined by perchloric acid precipitation and gel chromatography. Leakage of labeled molecules of different size indicated the size of the "functional pores" in the plasma membrane caused by the test substance. The nonionic detergent Triton X-100 produced large functional pores in the fibroblast membrane as evidenced by rapid leakage of both large and small labeled molecules. Theta-toxin from Clostridium perfringens and the polyene antibiotic filipin both gave rise to considerably small functional pores in the plasma membrane. Although small molecules easily passed the treated membrane, large molecules could not escape from the cells even after prolonged treatment with these substances or by increasing their concentration. By the contrast, the leakage profiles obtained with melittin from bee venom or with delta-toxin from Staphylococcus aureus in each case suggested the formation initially of pores of intermediate size that increased upon prolonged incubation or when higher concentrations were used.

Animals↗
PubMed1975

Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts.

A sensitive assay was developed for detection and quantitation of subtle permeability changes in the cytoplasmic membrane of human diploid fibroblasts. Release of the non-metabolizable amino acid [1-14C]alpha-aminoisobutyric acid (AIB; molecular weight (103) from the cytoplasm of prelabeled cells was used as an indicator of toxin-induced membrane damage. An optimal procedure for labeling these cells was designed after varying the conditions with regard to pH, temperature, concentration of AIB, composition of medium, and incubation time. Toxin-induced release of AIB was compared with release of a previously described nucleotide label, [3H]uridine. Melittin from bee venom and the polyene antibiotics filipin and amphotericin B in low concentrations induced a strikingly greater release of AIB than of nucleotide label. The sensitivity of this assay was furthermore demonstrated by treatment with the following bacterial cytolysins: phospholipase C and theta-toxin from Clostridium perfringens, alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus, and streptolysin S from Streptococcus pyogenes. In spite of their different modes of action, all these membrane-active toxins at low concentrations induced a significant release of AIB label. For an equal release of nucleotide label, several times higher concentrations were required.

Aminobutyrates↗
PubMed1973

Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: evaluation of a new quantitative as say for measuring cell damage.

Human diploid embryonic lung fibroblasts and HeLa cells were cultivated in Eagle minimaĺ essential medium supplemented with 10% calf serum. Monolayer cultures were labeled with (3)H-uridine and treated with highly purified staphylococcal alpha-, beta-, delta-, or gamma-hemolysin. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane after treatment with each hemolysin. The assay method described is simple, sensitive, and rapid. It allows quantitative estimation of changes in membrane permeability to be detected before a morphological damage is observed microscopically. Upon incubation for up to 30 min with highly purified staphylococcal hemolysins, only delta-hemolysin caused release of a significant amount of tritiated substances from fibroblasts. Such leakage occurred immediately after addition of delta-lysin and was independent of temperature. With minor exceptions, this was similar to the release of isotopes after treatment of the cells with the nonionic detergent Triton X-100. Treatment of fibroblasts with combinations of two or three of these toxins gave neither a synergistic nor an antagonistic effect. Evidence is presented which indicates that delta-hemolysin is the only important fibroblast damaging activity in crude preparations of extracellular proteins of four strains of S. aureus, whereas HeLa cells are susceptible also to purified alpha-toxin.

Cell Line↗
PubMedDate not supplied

The three-dimensional structure of trypsin-treated Staphylococcus aureus alpha-toxin.

Trypsin treatment of staphylococcal alpha-toxin cleaves the molecule into two roughly equally sized parts, which results in inactivation of the toxin. Tetragonal arrays of oligomers, closely resembling the native ones, can however be formed on lipid layers. From tilted views of negatively stained crystals a 3D structure to 23 A resolution has been determined by electron microscopy and image processing. On comparison with the 3D structure of the native alpha-toxin (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990) the subdomains are more separated, confirming the differences found when comparing the projection maps (Olofsson et al., J. Struct. Biol. 106, 199-204, 1991). The tryptic cleavage takes place in a postulated hinge region. The results are consistent with the hypothesis that the conformational change required for inducing the membrane permeabilizing property takes place in this region. Furthermore, we present a refined projection map at approximately 10 A resolution based on the analysis of a large number of crystals using unbending methods.

Bacterial Toxins↗