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PMID: 169201 Published · ppublish English Journal Article

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

Infection and immunity ·Vol. 12 ·No. 2 ·1975-08-00 ·Pages 225-32

Thelestam M, Möllby R

Abstract

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.

MeSH Terms
Aminobutyrates/metabolism Amphotericin B/pharmacology Cell Membrane Permeability/drug effects Clostridium perfringens/enzymology Cytoplasm/metabolism Diploidy Enterotoxins/pharmacology Escherichia coli Fibroblasts/drug effects Filipin/pharmacology Humans Melitten/pharmacology Phospholipases/pharmacology Polyethylene Glycols/pharmacology Staphylococcus Streptococcus pyogenes Streptolysins/pharmacology Toxins, Biological/pharmacology Uridine/metabolism
Chemicals
Aminobutyrates Enterotoxins Streptolysins Toxins, Biological Melitten Polyethylene Glycols Amphotericin B Filipin Phospholipases Uridine
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Thelestam M
Möllby R
References (37)
37 references, click to expand
  1. Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts.
    Infect Immun. 1975 Apr;11(4):640-8 PMID: 164404
  2. The identification and purification of multiple forms of theta-haemolysin (theta-toxin) of Clostridium perfringens type A.
    J Gen Microbiol. 1975 Apr;87(2):219-38 PMID: 167102
  3. Histamine release from mast cells by highly purified phospholipase C (alpha-toxin) and theta-toxin from Clostridium perfringens.
    Toxicon. 1974 Mar;12(2):199-208 PMID: 4136773
  4. Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes.
    Cell Immunol. 1974 Nov;14(2):284-302 PMID: 4219591
  5. Transport of amino acids by confluent and nonconfluent 3T3 and polyoma virus-transformed 3T3 cells growing on glass cover slips.
    J Biol Chem. 1969 May 25;244(10):2675-81 PMID: 4306034
  6. The uptake of non-metabolizable amino acids as an index of cell viability in vitro.
    Exp Cell Res. 1970 Aug;61(2):235-45 PMID: 4318927
  7. Molecular sieving of red cell membranes during gradual osmotic hemolysis.
    J Membr Biol. 1972 Dec 29;10(3):345-56 PMID: 4356143
  8. Purification of phospholipase C (alpha-toxin) from Clostridium perfringens.
    Biochim Biophys Acta. 1973 Oct 10;321(2):569-84 PMID: 4357667
  9. Interruption of the sequential release of small and large molecules from tumor cells by low temperature during cytolysis mediated by immune T-cells or complement.
    Proc Natl Acad Sci U S A. 1974 Jan;71(1):177-81 PMID: 4359327
  10. Action of Escherichia coli enterotoxin: adenylate cyclase behavior of intestinal epithelial cells in culture.
    Infect Immun. 1974 Jun;9(6):1003-10 PMID: 4364505
  11. Effect of Clostridium perfringens phospholipase C(alpha-toxin) on the human diploid fibroblast membrane.
    J Membr Biol. 1974;16(4):313-30 PMID: 4366075
  12. Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics.
    Biochim Biophys Acta. 1974 Feb 26;339(1):30-43 PMID: 4546884
  13. The release of potassium ions from Candida albicans in the presence of polyene antibiotics.
    J Gen Microbiol. 1974 Feb;80(2):451-65 PMID: 4596987
  14. Characteristics of streptolysin O hemolysis: kinetics of hemoglobin and 86rubidium release.
    Infect Immun. 1974 Jun;9(6):1022-7 PMID: 4598253
  15. The mode of action of polyene antibiotics; induced potassium leakage in Candida albicans.
    J Gen Microbiol. 1974 Apr;81(2):325-30 PMID: 4599408
  16. Fungal sterols and the mode of action of the polyene antibiotics.
    Adv Appl Microbiol. 1974;17(0):109-34 PMID: 4607548
  17. Studies on the mechanism of lymphocyte-mediated cytolysis. II. The use of various target cell markers to study cytolytic events.
    J Immunol. 1973 Jan;110(1):73-84 PMID: 4631074
  18. The use of 51Cr release as a measure of lymphocyte-mediated cytolysis in vitro.
    Cell Immunol. 1973 Dec;9(3):474-6 PMID: 4758877
  19. 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.
    Infect Immun. 1973 Dec;8(6):938-46 PMID: 4784889
  20. Coupled transport of sodium and organic solutes.
    Physiol Rev. 1970 Oct;50(4):637-718 PMID: 4919599
  21. Uptake of alpha-aminoisobutyric acid by cultured human fibroblasts.
    Biochim Biophys Acta. 1970 Dec 1;219(2):500-2 PMID: 5497208
  22. Interaction of staphylococcal alpha-toxin with artificial and natural membranes.
    J Bacteriol. 1968 Mar;95(3):1153-68 PMID: 5643052
  23. The effects of varying the cellular and extracellular concentrations of sodium and potassium ions on the uptake of glycine by mouse ascites-tumour cells in the presence and absence of sodium cyanide.
    Biochem J. 1968 Jul;108(3):489-98 PMID: 5667259
  24. Modes of mediated exodus of amino acids from the Ehrlich ascites tumor cell.
    J Biol Chem. 1968 Oct 25;243(20):5428-38 PMID: 5702053
  25. Transport of glycine by hemolyzed and restored pigeon red blood cells. Symmetry properties, trans effects of sodium ion and glycine, and their description by a single rate equation.
    J Biol Chem. 1968 Dec 10;243(23):6140-50 PMID: 5723458
  26. The kinetics of release of 86rubidium and hemoglobin from erythrocytes damaged by antibody and complement.
    Immunochemistry. 1969 Mar;6(2):295-307 PMID: 5778781
  27. Interaction of alytic polypeptide, melittin, with lipid membrane systems.
    J Biol Chem. 1969 Jul 10;244(13):3575-82 PMID: 5794226
  28. On the nature of the "non-saturable" migration of amino acids into Ehrlich cells and into rat jejunum.
    Bibl Laeger. 1966 Mar 14;112(3):524-31 PMID: 5912020
  29. Membranes of animal cells. II. The metabolism and turnover of the surface membrane.
    J Cell Biol. 1968 Jun;37(3):729-46 PMID: 11905204
  30. Metabolism in the rat of three amino acids lacking alpha-hydrogen.
    J Biol Chem. 1956 May;220(1):287-94 PMID: 13319346
  31. The cytotoxic action of immune gamma globulin and complement on Krebs ascites tumor cells. II. Chemical studies.
    J Exp Med. 1959 May 1;109(5):511-21 PMID: 13641574
  32. Amino acid metabolism in mammalian cell cultures.
    Science. 1959 Aug 21;130(3373):432-7 PMID: 13675766
  33. Studies of cytotoxicity using P-32.
    Aust J Exp Biol Med Sci. 1963 Jun;41:255-64 PMID: 13958679
  34. HEMOLYSIS OF RABBIT ERYTHROCYTES BY PURIFIED STAPHYLOCOCCAL ALPHA-TOXIN. III. POTASSIUM RELEASE.
    J Bacteriol. 1964 Jan;87:145-9 PMID: 14102847
  35. ACTION OF STAPHYLOCOCCAL TOXIN ON HUMAN PLATELETS.
    J Infect Dis. 1964 Dec;114:488-502 PMID: 14233140
  36. QUANTITATIVE TITRATIONS OF MOUSE H-2 ANTIBODIES USING CR-51-LABELLED TARGET CELLS.
    Transplantation. 1965 May;3:423-31 PMID: 14285093
  37. Inhibition of amino Acid transport in rat kidney cortex by puromycin.
    Proc Natl Acad Sci U S A. 1967 Feb;57(2):371-8 PMID: 16591480
Article Info
Journal
Infection and immunity
Abbr.
Infect Immun
ISSN
0019-9567
DOI
10.1128/iai.12.2.225-232.1975
Published
1975-08-00
Pages
225-32
Language
English
Region
United States
NLM ID
0246127
PMCID
PMC415272
Subset
IM
External Links
PubMed source DOI: 10.1128/iai.12.2.225-232.1975 PMC Full Text View journal details
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