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S Olsnes

Publications and source records attributed to S Olsnes.

At least 91 records · Page 5Linked to original sources
PubMed1989

Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non-toxic mutant proteins synthesized in vitro. Proteins obtained by N-terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N-terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C-terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment-derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N-terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease-inaccessible polypeptide of 18 kd (P18) was observed.

Animals↗
PubMed1989

Permeabilization of the plasma membrane by deletion mutants of diphtheria toxin.

Diphtheria toxin B-fragment binds to cell-surface receptors and facilitates translocation of the enzymatically active A-fragment to the cytosol. In this process the B-fragment inserts into the plasma membrane and induces formation of cation-selective channels. We examined the ability of a number of diphtheria toxin-derived molecules translated in vitro to permeabilize cells. Two proteins consisting of the whole B-fragment and small parts of the A-fragment, and one protein comprising most of the B-fragment alone, were more efficient than full-length toxin in permeabilizing the plasma membrane to monovalent cations. Two shorter B-fragment-derived proteins, with 3 and 10 kd N-terminal deletions, permeabilized the cells to sulfate and sucrose in addition to monovalent cations. The relationship between channel formation and toxin translocation is discussed.

Animals↗
PubMed1989

Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells.

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 micrograms/ml) for 1 h at 37 degrees C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-micron secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.

Antibodies, Monoclonal↗
PubMed1989

Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1.

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa S3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0 degrees C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37 degrees C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits.

Ammonium Chloride↗
PubMed1989

Immunotoxins--entry into cells and mechanisms of action.

Immunotoxins, that is conjugates of enzymatically active toxins with antibodies against cell surface antigens, are being used in targeted cell destruction. The directed toxin enters the cytosol of the target cells and inactivates components of the protein synthesis machinery. In this review, Sjur Olsnes, Kirsten Sandvig, Ole W. Petersen and Bo van Deurs discuss the mechanism of action of such conjugates and how they enter the cytosol. Emphasis is placed on points that should be considered in the preparation of efficient conjugates.

Humans↗
PubMed1989

Effect of anti-inflammatory analgesic drugs on the regulation of cytosolic pH by anion antiport.

Four nonsteroidal anti-inflammatory analgesic drugs acetylsalicylic acid (aspirin), salicylic acid, indomethacin and piroxicam were tested for their action on chloride/bicarbonate exchange by anion antiport and on the regulation of cytosolic pH (pHi) in Vero cells. The Na+-independent Cl-/HCO3- exchange regulates pHi back to normal after alkalinization and is therefore in a state of high activity at alkaline pHi, whereas it is in a state of low activity when pHi is below neutrality. Preincubation with the drugs at low pH strongly increased the activity of the Na+-independent Cl-/HCO3- exchange, whereas at high pH the antiport was inhibited. The Na+-dependent Cl-/HCO3- exchange, which is most active at low pHi and normalize pHi after acidification, was inhibited after preincubation with the drugs. In cells that had been incubated with the drugs, pHi was 0.1 to 0.4 pH units lower than in cells that had been incubated in the absence of drugs. The prostaglandins E2, F2 and I2 had little effect on anion antiport, and did not counteract the effects of the anti-inflammatory drugs on Cl-/HCO3- exchange, indicating that these actions of the drugs are not due to the inhibition of prostaglandin synthesis. The relevance of our findings to the clinical effects of these drugs is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

[Endocytosis, intracellular transport and sorting of proteins].

A complicated interaction between outgoing and incoming protein transport occurs in eucaryotic cells. The incoming protein transport commences when protein molecules (ligands) cleave to specific glycoprotein- or glycolipid-receptors on the cell surfaces for endocytosis thereby. This can proceed in two types of membrane vesicle. Endocytosed ligands and receptors are transported from the surface of the cell to its endosomal system where a dissociation of the ligand from its receptor and sorting with regard to its next destination may occur. There are a good many possibilities of intracellular transport, depending on which ligand/receptor complex is at issue. Thus ligands and receptors can be transported back to the cell surface (recycling), across e.g. a polarized epithelial cell (transcytosis) to the lysosomes, and to the cell's Golgi apparatus. Apart from a large number of physiological ligands, viruses and diverse bacterial and plant toxins are also endocytosed. This allows scope for a therapeutic use of endocytosis.

Animals↗
PubMed1988

Requirement of specific receptors for efficient translocation of diphtheria toxin A fragment across the plasma membrane.

The role of specific receptors in the translocation of diphtheria toxin A fragment to the cytosol and for the insertion of the B fragment into the cell membrane was studied. To induce nonspecific binding to cells, toxin was either added at low pH, or biotinylated toxin was added at neutral pH to cells that had been treated with avidin. In both cases large amounts of diphtheria toxin became associated with the cells, but there was no increase in the toxic effect. There was also no increase in the amount of A fragment that was translocated to the cytosol, as estimated from protection against externally added Pronase E. In cells where specific binding was abolished by treatment with 12-O-tetradecanoyl-phorbol 13-acetate, trypsin, or 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, unspecific binding did not induce intoxication or protection against protease. This was also the case in untreated L cells, which showed no specific binding of the toxin. When Vero cells with diphtheria toxin bound to specific receptors were exposed to low pH, the cells were permeabilized to K+, whereas this was not the case when the toxin was bound nonspecifically at low pH or via avidin-biotin. The data indicate that the cell-surface receptor for diphtheria toxin facilitates both insertion of the B fragment into the cell membrane and translocation of the A fragment to the cytosol.

Animals↗
PubMed1988

Diphtheria toxin-induced channels in Vero cells selective for monovalent cations.

Ion fluxes associated with translocation of diphtheria toxin across the surface membrane of Vero cells were studied. When cells with surface-bound toxin were exposed to low pH to induce toxin entry, the cells became permeable to Na+, K+, H+, choline+, and glucosamine+. There was no increased permeability to Cl-, SO4(-2), glucose, or sucrose, whereas the uptake of 45Ca2+ was slightly increased. The influx of Ca2+, which appears to be different from that of monovalent cations, was reduced by several inhibitors of anion transport and by verapamil, Mn2+, Co2+, and Ca2+, but not by Mg2+. The toxin-induced fluxes of N+, K+, and protons were inhibited by Cd2+. Cd2+ also protected the cells against intoxication by diphtheria toxin, suggesting that the open cation-selective channel is required for toxin translocation. The involvement of the toxin receptor is discussed.

Animals↗
PubMed1988

Role of chloride/bicarbonate antiport in the control of cytosolic pH. Cell-line differences in activity and regulation of antiport.

Sodium-linked and sodium-independent HCO3-/Cl- antiport was measured under different conditions in a number of cell lines. Transport of HCO3- was estimated from its effect on intracellular pH (pHi) measured with the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. The associated ion fluxes were estimated from the transport of 36Cl- and 22Na+. Na+-dependent and Na+-independent HCO3-/Cl- antiport were found in many, but not in all cell lines tested. The Na+-independent HCO3-/Cl- antiport was found to be highly pHi-dependent in a number of cell lines, whereas in others this was not the case. Some cell lines were found to have both Na+-dependent and Na+-independent HCO3-/Cl- antiport, whereas in others we could detect only one of these mechanisms. Na+/H+ antiport, which is quantitatively the most important H+-extruding mechanism, was found in all cell lines tested, but the activity varied strongly. Possible reasons for the qualitative and quantitative differences in antiport activity are discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Low pH-induced release of diphtheria toxin A-fragment in Vero cells. Biochemical evidence for transfer to the cytosol.

When Vero cells with surface-bound 125I-labeled, nicked diphtheria toxin were exposed to pH 4.5, two polypeptides of Mr 20,000 and 25,000 became protected against externally applied Pronase E. The 20-kDa polypeptide appears to be the toxin A-fragment, whereas the 25-kDa polypeptide must be derived from the B-fragment. Permeabilization of the cells with saponin allowed efflux of the 20-kDa fragment to occur, whereas most of the 25-kDa polypeptide remained associated with the cells. A number of compounds and conditions which protect cells against diphtheria toxin prevented the protection against Pronase E. Protection of the 25-kDa polypeptide occurred even when the transmembrane proton gradient (delta pH) was dissipated by acidification of the cytosol, whereas protection and release of the A-fragment were prevented under these conditions. Electrical depolarization and ATP depletion of the cells did not inhibit protection and release of the A-fragment. The data indicate that delta pH is required for the transfer of the A-fragment to the cytosol, whereas the insertion of part of the B-fragment into the membrane occurs at low pH, even in the absence of a delta pH.

Adenosine Diphosphate Ribose↗
PubMed1988

Inhibition of endocytosis from coated pits by acidification of the cytosol.

Binding and endocytosis of the ligands transferrin, epidermal growth factor (EGF), and ricin were measured in a number of different cell lines after treatment of cells with compounds that react with SH-groups and under conditions where the cytosolic pH was lowered. N-ethylmalemide and diamide irreversibly inhibited endocytosis of all ligands tested, whereas low pH in the cytosol strongly inhibited endocytosis of transferrin and EGF. Data obtained by electron microscopy indicated that the formation of coated vesicles from coated pits is inhibited in acidified cells. Entry of ricin was much less affected, and ricin endocytosed under these conditions was able to intoxicate the cells. At low pH in the cytosol there was a calcium-dependent increase in the number of transferrin receptors at the cell surface. The increase was even larger in the presence of the calcium ionophore A23187, whereas it was completely blocked by the calmodulin antagonists trifluoperazine and W7. The results show that endocytosis from coated pits can be inhibited in a reversible way by acidification of the cytosol and they suggest that a second pathway of endocytosis exists, possibly involving formation of vesicles from uncoated areas of the membrane.

Ammonium Chloride↗
PubMed1988

Estimation of the amount of internalized ricin that reaches the trans-Golgi network.

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

Animals↗
PubMed1988

Characterization of a toxic lectin in Iscador, a mistletoe preparation with alleged cancerostatic properties.

Iscador, a mistletoe preparation widely used in cancer chemotherapy, was found to contain a toxic component which is structurally and functionally closely related to viscumin. When antiviscumin was present in the medium, Vero cells were strongly, but not completely protected against Iscador. The main cytotoxic component in Iscador appears, like viscumin, to bind to terminal nonreducing galactose residues. Thus, addition to the medium of galactose, lactose or melibiose blocked the cytotoxic activity. The main cytotoxic protein in Iscador was retained on a desialylated fetuin column and on an antiviscumin column. This material was found to have a molecular weight close to, but not identical to, that of viscumin.

Animals↗