Biomaterial Database

Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. 1975

M Thelestam, and R Möllby

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.

UI	MeSH Term	Description	Entries
D008555	Melitten	Basic polypeptide from the venom of the honey bee (Apis mellifera). It contains 26 amino acids, has cytolytic properties, causes contracture of muscle, releases histamine, and disrupts surface tension, probably due to lysis of cell and mitochondrial membranes.	Melittin,Mellitin
D010740	Phospholipases	A class of enzymes that catalyze the hydrolysis of phosphoglycerides or glycerophosphatidates. EC 3.1.-.	Lecithinases,Lecithinase,Phospholipase
D011092	Polyethylene Glycols	Polymers of ETHYLENE OXIDE and water, and their ethers. They vary in consistency from liquid to solid depending on the molecular weight indicated by a number following the name. They are used as SURFACTANTS, dispersing agents, solvents, ointment and suppository bases, vehicles, and tablet excipients. Some specific groups are NONOXYNOLS, OCTOXYNOLS, and POLOXAMERS.	Macrogols,Polyoxyethylenes,Carbowax,Macrogol,Polyethylene Glycol,Polyethylene Oxide,Polyethyleneoxide,Polyglycol,Glycol, Polyethylene,Glycols, Polyethylene,Oxide, Polyethylene,Oxides, Polyethylene,Polyethylene Oxides,Polyethyleneoxides,Polyglycols,Polyoxyethylene
D002463	Cell Membrane Permeability	A quality of cell membranes which permits the passage of solvents and solutes into and out of cells.	Permeability, Cell Membrane
D003016	Clostridium perfringens	The most common etiologic agent of GAS GANGRENE. It is differentiable into several distinct types based on the distribution of twelve different toxins.	Clostridium welchii
D003593	Cytoplasm	The part of a cell that contains the CYTOSOL and small structures excluding the CELL NUCLEUS; MITOCHONDRIA; and large VACUOLES. (Glick, Glossary of Biochemistry and Molecular Biology, 1990)	Protoplasm,Cytoplasms,Protoplasms
D004171	Diploidy	The chromosomal constitution of cells, in which each type of CHROMOSOME is represented twice. Symbol: 2N or 2X.	Diploid,Diploid Cell,Cell, Diploid,Cells, Diploid,Diploid Cells,Diploidies,Diploids
D004768	Enterotoxins	Substances that are toxic to the intestinal tract causing vomiting, diarrhea, etc.; most common enterotoxins are produced by bacteria.	Staphylococcal Enterotoxin,Enterotoxin,Staphylococcal Enterotoxins,Enterotoxin, Staphylococcal,Enterotoxins, Staphylococcal
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005347	Fibroblasts	Connective tissue cells which secrete an extracellular matrix rich in collagen and other macromolecules.	Fibroblast