Biomedical subjects

M Thelestam

Publications and source records attributed to M Thelestam.

At least 73 records · Page 4Linked to original sources

PubMed1986

Early events in the action of staphylococcal alpha-toxin on the plasma membrane of adrenocortical Y1 tumor cells.

The early events in staphylococcal alpha-toxin action on mouse adrenocortical (Y1) tumor cells were studied. Cell-bound toxin could be partially neutralized by anti-alpha-toxin and inactivated by trypsin added within 10 min at 37 degrees C after the end of the binding step. Likewise, cell-bound toxin was capable of lysing rabbit erythrocytes (RRBC) added to the cells within 10 min after binding at 37 degrees C. After this time, the Y1 cells could not be rescued from intoxication by antibodies or trypsin, and the toxin was not accessible for lysis of RRBC. However, at 0 to 4 degrees C, the cell-bound toxin remained accessible to antibodies for at least 4 h. CaCl2 (30 mM) did not affect binding of the toxin to Y1 cells but completely prevented the intoxication if added within 10 min at 37 degrees C after the end of the binding step. The intoxication was independent of metabolic energy, active receptor clustering on the cell surface, and endocytosis of the toxin. Therefore, alpha-toxin interacted with the Y1 cell membrane in at least three separable steps: binding, a conformational change at the cell surface, and membrane damage. These early events appear to be similar to those occurring on RRBC treated with alpha-toxin.

Adrenal Cortex Neoplasms↗

PubMed1986

A staphylococcal alpha-toxin fragment. Its characterization and use for mapping biologically-active regions of alpha-toxin.

A fragment (alpha-13) of Staphylococcal alpha-toxin was compared with intact alpha-toxin as regards biochemical and biological properties, and the resulting information was used for mapping biologically-active regions of alpha-toxin. The alpha-13 fragment had an apparent Mr of 18,500, judged by sodium dodecylsulphate polyacrylamide gel electrophoresis. However, it showed the same relative mobility as alpha-toxin when subjected to gel filtration on Biogel P60, high pressure liquid chromatography or electrophoresis on polyacrylamide gradient gel. The fragment had roughly the same specific hemolytic activity as intact alpha-toxin. In contrast to intact alpha-toxin, the fragment was neither membrane-damaging to mouse adrenocortical (Y 1) tumour cells nor lethal to mice. However, a short treatment of Y 1 cells with the fragment completely blocked intoxication by subsequently-added alpha-toxin. Likewise, the lethal effect of alpha-toxin was inhibited when the fragment was injected prior to the toxin. Thus, the fragment had lost the active region(s) responsible for Y 1 cell intoxication and lethality, while the region(s) for binding to these targets, as well as the region responsible for hemolysis, were retained. On the basis of these findings and previous reports concerning tryptic fragments of alpha-toxin, a hypothetical map of the different biologically-active regions of alpha-toxin was established.

Animals↗

PubMed1985

Effect of thiol-activated toxins (streptolysin O, alveolysin, and theta toxin) on the generation of leukotrienes and leukotriene-inducing and -metabolizing enzymes from human polymorphonuclear granulocytes.

The generation of leukotrienes (LTC4, LTD4, LTE4, and LTB4; 12-epi-LTB4 isomer) from human granulocytes by thiol-activated toxins (streptolysin O, alveolysin from Bacillus alvei, and theta toxin from Clostridium perfringens) is described. The release occurs under noncytolytic conditions. Although LTB4 is the major component after calcium ionophore stimulation, more LTC4 as compared with LTB4 is released with the toxins. The 5-lipoxygenase pathway of toxin-mediated activation can effectively be inhibited by caffeic acid, a lipoxygenase inhibitor. The toxins also induce the release of leukotriene-metabolizing enzymes such as gamma-glutamyltranspeptidase, which transfers LTC4 into LTD4, and dipeptidase, which metabolizes LTD4, into LTE4. Dipeptidase activity is more pronounced than the gamma-glutamyltranspeptidase activity but still does not reach the levels obtained when cells were triggered with opsonized zymosan.

Arachidonate Lipoxygenases↗

PubMed1984

Polyphosphate-mediated protection from cellular intoxication with Clostridium difficile toxin B.

The influence of polyphosphorylated compounds on intoxication of human lung fibroblasts with Clostridium difficile toxin B was studied. ATP, as well as other nucleoside di-, tri-, and tetraphosphates, inorganic polyphosphates and polyphosphorylated sugars, caused a dose-dependent (1-5 mM range) delay in the appearance of the cytopathogenic effect. With a longer phosphate chain, the delay was more pronounced, although the cytopathogenic effect always developed finally, reaching the level of the control within 20 h. Toxin preparations contained one fraction of molecules able to bind ATP, besides one non-binding fraction. The protective effect of ATP did not depend on its energy producing ability. Neither was the protective effect due to an inactivation of the toxin per se, or to an interference with binding of the toxin to the cells. ATP was protective even upon addition 10 min after the toxin binding step. In the presence of ATP, the toxin remained accessible to neutralization with antitoxin. In analogy with the P-site on diphtheria toxin, we postulate that C. difficile toxin B contains a polyphosphate-binding site. This site is separate from the receptor-binding site, but involved in the interaction of toxin B with the cell surface shortly after the binding step.

Adenosine Triphosphate↗

PubMed1984

Evaluation of the biological activity of cigarette-smoke condensate fractions using six in vitro short-term tests.

The biological activity of the volatile part of the particulate phase of cigarette-smoke condensate, the semivolatile fraction, has been examined, since the constituents of this material are accessible to selective filtration. Such a process offers a possibility to reduce the biological activity of total cigarette smoke without appreciably affecting the taste. Cigarette-smoke condensate, obtained from domestic American blend type cigarettes, was therefore separated into a nonvolatile and a semivolatile fraction, and the latter was fractionated by liquid-liquid extractions into four subfractions; acids, phenols, bases, and neutrals. The biological activity of these fractions was investigated using six in vitro short-term tests, of which two, the Ames test and the induction of sister chromatid exchanges, provided information on their genotoxicity, and the other four provided information on their cytotoxicity by measuring inhibition of cell growth, inhibition of oxidative metabolism, membrane damage, and ciliotoxicity. Sister chromatid exchanges were found to be induced by the total condensate, the nonvolatile and the semivolatile fractions, and the subfractions derived from the semivolatile fraction, except the bases. The Ames test showed the total condensate and the nonvolatile fraction to contain direct-acting base-pair mutagens as well as indirect-acting frameshift mutagens. While the semivolatile fraction was found nonmutagenic, two of its subfractions, acids and phenols, were shown to contain base-pair mutagens, which did not require metabolic activation. The total condensate and the nonvolatile and semivolatile fractions showed similar activity in the four cytotoxicity tests. Of the semivolatile subfractions, the acids and the phenols exhibited the highest activity and the bases the lowest; the toxicity observed for the neutrals varied with the test system used.

Animals↗

PubMed1984

Hydrophobicity of cultured mammalian cells and some effects of bacterial phospholipases C.

Eucaryotic cell surface hydrophobicity was measured as a partition of palmitic acid between the cell surface and the surrounding buffer. The method was found to be independent of cell mass or amount of palmitic acid within a large interval. An estimation of cell stability could also be obtained. The effects of Ca and Mg ions on cell hydrophobicity and stability of mouse myeloma (SP2/O) cells and of Chinese hamster ovary (CHO) cells were determined. This system permits measurement of changes in cell hydrophobicity caused by various additives, e.g. ions, purified bacterial products, antibiotics or cytostatics. Studies were made on these eucaryotic cells treated with purified bacterial phospholipases C from S. aureus and C. perfringens. These enzymes were found to increase the eucaryotic cell membrane hydrophobicity. This finding might indicate that bacterial phospholipases C facilitate bacterial colonization at the start of an infection.

Animals↗

PubMed1983

Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts.

In cultured human lung fibroblasts treated with Clostridium difficile cytotoxin, the latency before appearance of the cytopathogenic effect was dose-related with a minimum of 45 min. At 37 degrees C, the toxin was accessible on all cells to inactivation with trypsin or neutralization with antitoxin during the first tenth of the latency. At 0 degrees C, the toxin was accessible considerably longer. The cytopathogenic effect was reversibly prevented by the lysosomotropic agents chloroquine and ammonium chloride, which had to be added within one-fifth of the latency to protect all cells. In the presence of chloroquine, but not of ammonium chloride, the time period during which the toxin remained amenable to neutralization with antitoxin was prolonged. The protective effect of ammonium chloride was not influenced by dropping the extracellular pH to 4.5, but that of chloroquine was abolished. The expression of the intoxication was not affected by inhibitors of the DNA, RNA or protein synthesis. Inhibitors of the energy metabolism prevented the cytopathogenic effect when added before the last phase of the latency. The results suggest that expression of the cytopathogenic effect requires internalization of the toxin, and that metabolic energy but no macromolecular synthesis is needed for the action of the toxin after this internalization.

Bacterial Proteins↗

PubMed1983

Membrane damage by staphylococcal alpha-toxin to different types of cultured mammalian cell.

Staphylococcal alpha-toxin was shown to be more membrane-damaging to epithelial-like cells than to neuroblasts or normal fibroblasts. Mouse adrenal cortex tumor (Y1Ac) epithelioid cells and human embryonal lung (MRC-5) fibroblasts were used for further comparison. Alpha-toxin was considerably more cytotoxic to adrenal cells than to fibroblasts. This difference did not depend on the presence fibronectin on the fibroblast surface, or on a general difference in the response to other membrane-damaging hemolytic toxins and detergents. Incubation of adrenal cells at 0 degree C with alpha-toxin induced some irreversible change, and membrane damage and a cytotoxic effect developed upon further incubation in toxin-free growth medium. In fibroblasts the membrane damage progressed slowly and only in the continued presence of the toxin. Toxin-induced damage to transport and synthetic functions in fibroblasts was reversible upon removal of the toxin after prolonged exposure. It is proposed that adrenal cells may carry a cell-surface receptor to which alpha-toxin binds specifically, thereby allowing the toxin to exert its cell damaging effect.

Animals↗

PubMed1983

Photolabeling of staphylococcal alpha-toxin from within rabbit erythrocyte membranes.

Intrinsic membrane proteins of rabbit red blood cells were labeled with the photoreactive amphipatic reagent 12-(4-azido-2-nitrophenoxy) stearoyl (1-14C) glucosamine, which inserts into the hydrophobic membrane region and generates a reactive nitrene upon ultraviolet irradiation. Photolabeling of membrane-bound staphylococcal alpha-toxin after lysis of probe-treated rabbit red blood cells by this toxin implies its penetration into the hydrophobic region of the outer leaflet of the membrane. In contrast clostridial theta-toxin and staphylococcal delta-toxin were not labeled, but extraction of intrinsic membrane proteins by delta-toxin was evidenced.

Animals↗

PubMed1983

Survival of cultured cells after functional and structural disorganization of plasma membrane by bacterial haemolysins and phospholipases.

Lesions were induced in the plasma membranes of cultured human fibroblasts by membrane damaging toxins of bacterial origin (haemolysins). Structural disorganization of the membrane was measured as leakage of a radiolabelled small cytoplasmic marker and functional membrane damage was measured as decreased uptake of aminoisobutyrate. Cell survival was scored 24 and 48 hr later by measuring uptake of Trypan Blue and by light microscopical evaluation of cell morphology and proliferation. The membrane damage induced by most bacterial toxins was reversible upon removal of the toxin, since toxin-treated cells recovered and excluded Trypan Blue although they had been permeable to the dye immediately after the toxin treatment. Among ten bacterial toxins tested, the only exception of this general behavior was the Aeromonas hydrophila beta-haemolysin, which irreversibly damaged human fibroblasts. Thus, the action of bacterial haemolysins on cultured cells generally seems restricted to a plasma membrane permeabilization, which is reversible regardless of the mechanism of membrane damaging action of the toxin or of the relative size of the structural lesions induced. Furthermore, the use of Trypan Blue uptake as a measure of cell death caused by membrane damaging agents appears to be of limited value.

Aeromonas↗

PubMed1983

Loss of surface fibronectin from human lung fibroblasts exposed to cytotoxin from Clostridium difficile.

Clostridium difficile cytotoxin caused an irreversible dose- and time-dependent loss of fibronectin from the surfaces of human lung fibroblasts, paralleling the appearance of the cytopathic effect. Fibronectin was not required for the intoxication process. The results lend further support to a transmembrane connective link between fibronectin and the microfilaments.

Bacterial Proteins↗

PubMed1982

Two membrane damaging toxins from Aeromonas hydrophila.

The effects of two hemolysins (alpha and beta) from Aeromonas hydrophila on human lung fibroblasts were investigated. The toxins differed with respect to relative size of lesions induced in the plasma membrane, morphological effects, reversibility of effects and adsorption to fibroblasts. The effect of alpha-hemolysin appears to be restricted to the plasma membrane level, whereas the beta-hemolysin is strongly cytotoxic and probably acts on some intracellular target in addition to the primary target, the plasma membrane.

Aeromonas↗

PubMed1982

Two different modes of membrane damaging action by bacterial thiol-activated haemolysins.

The mode of action to cause membrane damage to human diploid fibroblasts by streptolysin 0 is not typical for that of all thiol-activated cytolysins. Theta-toxin and alveolysin, on the other hand, share all properties investigated and may thus, with regard to mechanisms of membrane damaging action, constitute a subgroup among thiol-activated haemolysins.

Adsorption↗

PubMed1981

Membrane-damaging action of alveolysin from Bacillus alvei.

We investigated membrane damage to human diploid, embryonic lung fibroblasts caused by highly purified alveolysin by measuring leakage of cytoplasmic markers and uptake of various metabolites, and we observed a leakage pattern typical of SH-activated cytolysins. However, the mode of membrane interaction resembled the mode of membrane interaction of theta-toxin from Clostridium perfringens rather than that of streptolysin O in the following respects: the activity on fibroblast membranes was high compared with the activity on sheep erythrocytes; the toxin did not bind irreversibly to fibroblast cytoplasmic membranes; considerable membrane damage was caused at 0 degrees C; and inhibition of amino acid uptake occurred in the absence of significant structural membrane damage. These findings imply that data on membrane effects caused by streptolysin O cannot be related indiscriminately to other SH-activated cytolysins. With regard to the mode of membrane interaction, two apparently different groups of SH-activated cytolysins exist.

Bacillus↗

PubMed1981

Intoxication of cultured human lung fibroblasts with Clostridium difficile toxin.

The cytopathogenic effect of partially purified toxin from Clostridium difficile on cultured human lung fibroblasts was studied. Conditions for determination of 50% tissue culture dose were standardized. The cytopathogenic effect of the toxin was dependent on toxin concentration, exposure time, and density of the cells. Transfer of the cells to 0 degrees C did not inhibit binding of toxin to the fibroblast surface, but prevented the development of the cytopathogenic effect. Both binding of toxin and some intracellular step(s) were prevented by 2,4-dinitrophenol. These preventative effects were reversible. Before and concomitantly with the appearance of the cytopathogenic effect, the cellular uptake of uridine and of amino acids was markedly stimulated. Protein synthesis was depressed when 100% of the cells showed the cytopathogenic effect, but the synthesis of nucleic acids was inhibited only several hours later. The primary cellular target for the toxin is still unknown.

Bacterial Toxins↗

PubMed1981

Membrane-damaging and cytotoxic effects on human fibroblasts of alpha- and beta-hemolysins from Aeromonas hydrophila.

The effects of two hemolysins (alpha and beta) from Aeromonas hydrophila on human lung fibroblasts were investigated. The toxins differed distinctly in regard to the morphological changes they produced. The alpha-hemolysin caused rounding of the cells. The beta-hemolysin caused a striking vacuolization of the cytoplasm in cells which remained spread out on the growth surface. The toxins also differed as to relative size of the initial lesions they induced in the fibroblast membrane, scored by leakage of different-sized cytoplasmic markers. The alpha-hemolysin induced larger lesions than did the beta-hemolysin. It was indirectly demonstrated that the alpha-hemolysin did not bind, or bound only transiently, to the fibroblasts. By contrast, the beta-hemolysin bound rapidly and firmly. The cytopathogenic response to the alpha-hemolysin was reversible, whereas cells treated with small amounts of the beta-hemolysin for only 1 min invariably died within a few hours. Thus, the two hemolysins from A. hydrophila, despite many biochemical similarities, show essential dissimilarities in their interactions with cultured cells.

Aeromonas↗

PubMed1981

Toxinogenicity of clinical isolates of coagulase-negative staphylococci towards various animal cells.

The toxinogenicity of fifty coagulase-negative staphylococci isolated from various clinical syndromes was compared, using lysis of human erythrocytes, leakage of a radio-active marker from human embryonic lung fibroblasts by culture filtrates, and direct cytotoxicity of growing bacteria towards mouse skin fibroblasts in an agar overlay assay. Good correlation was obtained between those strains which elaborated greater than or equal to 16 HU/ml delta-toxin in culture, measurable also in terms of radioactive nucleotide leakage from tissue culture cells and those strains which caused a direct cytotoxicity effect in the colony overlay test. delta-toxin was implicated in the genesis of such cellular damage. Speciation of the coagulase-negative staphylococci revealed that isolates identified as S. epidermidis, S. saprophyticus and S. haemolyticus were most active in each of these tests. The colony overlay technique is suggested as being a potential screening assay for toxinogenic coagulase-negative staphylococci associated with clinical infections.

Animals↗

PubMed1980

Cultured human fibroblasts as a model for evaluation of potential in vivo toxicity of membrane damaging antibiotics.

The toxic side effects of certain antimicrobial agents are probably related to their membrane damaging properties. Thus it should be possible to use measurement of membrane damage in vitro for evaluation of the potential toxicity in vivo of such antibiotics. We estimated the membrane damage induced in cultured human fibroblasts by anti-microbial agents, such as polyene antibiotics, sodium fusidate and polymyxin B as well as derivatives of some of these. Degree and character of membrane damage was determined on basis of leakage of three defined cytoplasmic markers from prelabelled cells after treatment with test substance. By comparing the minimal inhibitory concentrations against the target microbial cells (MIC) with the amounts needed to cause membrane damage of human cells (ED50) a 'therapeutic dose range' was obtained (ED50/MIC). The therapeutic dose range and the character of induced membrane damage were compared with the relative toxicities in vivo of each test substance. Highly toxic agents caused large functional 'holes' and/or showed a narrow therapeutic dose range, whereas less toxic substances induced smaller functional holes and/or had a larger therapeutic dose range. These parameters, evaluated in the presented model system, should be useful for an indication of potential toxicity in vivo.

Aminoisobutyric Acids↗