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Biomedical subjects

S Olsnes

Publications and source records attributed to S Olsnes.

At least 163 records · Page 9Linked to original sources
PubMed1980

Isolation and characterization of Shigella shigae cytotoxin.

Shigella shigae cytotoxin was isolated from pressure-dialyzed culture medium and from a 26-year-old sample of partially purified toxin. The toxin was adsorbed to a column of acid-treated chitin at low salt concentration and eluted with 1 M NaCl. The partially purified toxin was labeled with 125 I and resubmitted to chromatography on acid-treated chitin. The labeled material eluted with 1 M NaCl was mixed with unlabeled rabbit hemoglobin as a carrier and the toxin was further purified by chromatography on a DE52 column and by sucrose gradient centrifugation. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the pure Shigella toxin migrated as two bands corresponding to molecular weights of 30,500 and of about 11,000. The intact toxin may consist of one heavy chain and four to five copies of the light chain. In isoelectric focusing experiments, Shigella toxin was recovered from a broad zone between pH 5.8 and pH 7.5. This appears to be due to charge heterogeneities both in the large and the small chain. Most cell lines tested were completely resistant even to high concentrations of Shigella toxin. Vero cells and one strain of HeLa cells were very sensitive, 2.5 pg/ml of pure toxin induced 50% inhibition of protein synthesis overnight in HeLa cells.

Animals↗
PubMed1980

A human melanoma cell line established from xenograft in athymic mice.

A human melanoma cell line with unusually high growth potential was established from a xenograft growing in athymic mice. When xenograft fragments were cultured in vitro, melanoma cells grew out rapidly without any contamination of mouse stromal cells. An established cell line, FME, derived from this tumour, grew both in monolayer and in shaker suspension culture with doubling times of about 20 h. The cells grew easily at low serum concentrations and could even be cultured in serum-free medium supplemented with insulin and transferrin. The cultured cells were hyperdiploid, as were the cells of the xenograft. The cells grew easily in soft agar and formed tumours in athymic mice. When growing exponentially, the cells were almost unpigmented, but when grown to high density, their melanin content increased. Upon treatment with dimethyl sulphoxide (DMSO), retinoic acid and theophylline, as well as with the tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), the cells showed growth inhibition and increased melanin synthesis.

Animals↗
PubMed1980

Pathways involved in fluid phase and adsorptive endocytosis in neuroblastoma.

The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques. The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population. Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP. In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.

Adsorption↗
PubMed1980

Diphtheria toxin entry into cells is facilitated by low pH.

At neutral pH, NH4Cl and chloroquine protected cells against diphtheria toxin. A brief exposure of the cells to low pH (4.5-5.5) at 37 degrees completely abolished this protection. When, to cells preincubated with diphtheria toxin and NH4Cl, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished. A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH. Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained. The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell. The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes. Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin.

Abrin↗
PubMed1980

Interaction of Shigella shigae cytotoxin with receptors on sensitive and insensitive cells.

The effect of a cytotoxin isolated from Shigella shigae has been tested on different cell lines. HeLa S3 cells, as well as some other human carcinoma cells, were killed by picomolar to femtomolar concentrations of the pure toxin, whereas certain other human carcinoma cells and a variety of non-epithelial cells from human tissue and from various animal tissues were resistant to-nanomolar concentrations of the toxin. Binding studies with 125 I-labelled Shigella shigae cytotoxin showed that the sensitive HeLa S3 cells contain 1.3 x 10(6) binding sites per cell, whereas in an insensitive HeLa cell line 2.6 x 10(5) sites per cell were measured. In all cases the apparent association constant, Ká was found to be about 10(10) M-1. The binding occurred fairly rapidly, whereas dissociation of bound toxin occurred at a very slow rate, even in the presence of excess unlabaled toxin. All toxin sensitive cell lines bound similar amounts of toxin as HeLa S3 cells, whereas some of the resistant cell lines did not contain measurable amounts of toxin receptors.

Animals↗
PubMed1980

Human embryonal carcinoma grown in athymic mice and in vitro.

Tissue from 19 human testis tumors was transplanted into athymic mice. One embryonal carcinoma, ECCS, grew rapidly, and this tumor was studied both as a xenograft and an in vitro culture of xenograft-derived tumor cells. Xenografts showed no evidence of differentiation. The embryonal carcinoma cells were heteroploid and showed alkaline phosphatase activity. When tumor cells from the xenografts were grown in vitro, the cells formed aggregates resembling embryoid bodies with epithelium-like cells in the periphery. Regularly, another population of mouse cells which showed several criteria of malignancy overgrew the culture and could be subcultured continuously. These abnormal cells may result from an in vivo or in vitro transformation of mouse stromal cells.

Alkaline Phosphatase↗
PubMed1978

Properties and action mechanism of the toxic lectin modeccin: interaction with cell lines resistant to modeccin, abrin, and ricin.

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possess different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.

Carbohydrate Metabolism↗