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K Sandvig

Publications and source records attributed to K Sandvig.

At least 145 records · Page 8Linked to original sources
PubMed1985

Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin.

When Vero cells were depleted of potassium, the cells were protected against diphtheria toxin. Potassium depletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L-cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells to bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium.

ADP Ribose Transferases↗
PubMed1985

Tumor-promoting phorbol esters and vanadate alter the sensitivity of HeLa S3 cells to poliovirus type 1.

Treatment of HeLa S3 cells with tumor-promoting phorbol esters and vanadate increased their sensitivity to type 1 poliovirus. Since the sensitization could not be accounted for by increased virus binding or virus production, it appears that virus entry was facilitated by the treatments. When HeLa S3 cells were incubated with TPA for prolonged periods of time, they became resistant to poliovirus due to reduced ability to bind the virus.

Cell Membrane↗
PubMed1985

Receptor-mediated endocytosis of a ricin-colloidal gold conjugate in vero cells. Intracellular routing to vacuolar and tubulo-vesicular portions of the endosomal system.

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many "physiological' ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.

Animals↗
PubMed1985

Entry mechanisms of protein toxins and picornaviruses.

The mode of entry into cells of a number of protein toxins with intracellular sites of action and of three picornaviruses is discussed. Of the different toxins in this group, diphtheria toxin has been most thoroughly studied with respect to its uptake mechanism. This toxin binds to cell surface receptors which are possibly part of the major anion-transport system in the cells. The bound toxin is then endocytosed and, when the pH drops below pH 5, a normally hidden hydrophobic domain is exposed and inserted into the membrane. By a process which, in addition to low pH, requires chloride transport and a proton gradient across the membrane, the toxin A fragment is translocated to the cytosol. When diphtheria toxin is bound at the cell surface, rapid entry through the surface membrane can be induced by treatment with low pH. Modeccin and Pseudomonas exotoxin A also require low pH for entry, but low pH is not able to induce rapid entry of these toxins from the cell surface. Another group of toxins, abrin, ricin and viscumin, is characterized by the fact that low pH in the medium prevents the toxins from entering the cytosol, but not from entering endocytic vesicles. However, when the pH is subsequently returned to neutrality the endocytosed toxins are able to enter the cytosol. In the picornaviruses the entry of a single hydrophilic macromolecule per cell is also sufficient to induce maximal biological effect. Poliovirus, like diphtheria toxin, appears to enter the cytosol from an acidic intracellular compartment which may be the endosome. Also human rhinovirus 2 requires low pH for entry, whereas encephalomyocarditis virus does not enter at low pH. The similarities and differences between the uptake mechanisms of toxins and viruses are discussed.

ADP Ribose Transferases↗
PubMed1984

Dimethyl sulphoxide protects cells against polypeptide toxins and poliovirus.

The effect of dimethyl sulphoxide and other cryoprotective compounds on the sensitivity of cells to polypeptide toxins and to poliovirus was tested. In the presence of these compounds, which all affect membrane fluidity, the cells were protected against the toxic proteins and against poliovirus. The large protection obtained was not due to reduced binding and endocytosis of the toxins. Apparently, the cryoprotective compounds interfere with the entry of toxins and of the poliovirus genome across the cell membrane.

Abrin↗
PubMed1984

Requirements for entry of poliovirus RNA into cells at low pH.

HeLa S3 cells were protected against infection by poliovirus type I by the presence of monensin and N,N'-dicyclohexylcarbodiimide (DCCD), compounds elevating the pH of acidic intracellular compartments. The protection was fully overcome by exposing the cells to pH 5.5 and lower, and at approximately pH 6.1 it was reduced by half. Measurements of the ability of the virus to enter the detergent phase under conditions where Triton X-114 was separated from water indicated that the virus is hydrophilic at neutral pH, and that it exposes hydrophobic regions at low pH. When the cells were pretreated with acetic acid, which reduces the intracellular pH, virus entry was inhibited, indicating that a pH gradient across the membrane is necessary for infection. Under all conditions which induced infection, the virus particles were altered to more slowly sedimenting material. Also, virus bound to aldehyde-fixed cells was altered when exposed to low pH at 37 degrees C. The data indicate that poliovirus bound to receptors on cells exposes hydrophobic regions at low pH, and that at physiological temperature it undergoes alteration. This alteration may be a necessary, but not sufficient requirement for infection.

Carbodiimides↗
PubMed1984

Anion requirement and effect of anion transport inhibitors on the response of vero cells to diphtheria toxin and modeccin.

The anion requirement for toxic action of diphtheria toxin and modeccin was studied. In Cl- -free Hepes buffer made isotonic with mannitol, cells were insensitive to diphtheria toxin and modeccin. Just 2 mM NaCl was sufficient to obtain full toxic activity of modeccin, whereas 140 mM NaCl was required for maximal intoxication with diphtheria toxin. Br- could substitute for Cl-. NO3-,l-, and ClO3- were less efficient than Cl-, whereas SO42- and SCN- were unable to replace Cl- . Cl- deprivation both reduced the ability of cells to bind diphtheria toxin and prevented bound toxin from intoxicating the cells. The binding of modeccin was not reduced. SITS (4-acetamide-4'-isothiocyano-stilbene-2,2'-disulfonic acid), an inhibitor of Cl- entry, protected against diphtheria toxin and modeccin, indicating that Cl- transport is required for intoxication.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1984

Calmodulin antagonists sensitize cells to pseudomonas toxin.

L cells and mouse 3T3 cells, which are very sensitive to Pseudomonas aeruginosa exotoxin A (PEA), were protected with weak bases and low concentrations of monensin. BHK cells and a number of other cell lines which are much less sensitive to PEA were much less protected under these conditions. Trifluoperazine, dansylcadaverine, and several other calmodulin antagonists strongly sensitized BHK cells to the toxin whereas they did not affect the sensitivity of the mouse 3T3 and L cells. The sensitization of the BHK cells was counteracted by treatment with weak bases or low concentrations of monensin. Calmodulin antagonists also sensitized cells to toxin which had become inaccessible to antitoxin, indicating that the effect of the calmodulin antagonists is exerted on a process taking place after the toxin is endocytosed.

ADP Ribose Transferases↗
PubMed1984

Different pH requirements for entry of the two picornaviruses, human rhinovirus 2 and murine encephalomyocarditis virus.

The entry into cells of human rhinovirus 2 (HRV 2) and murine encephalomyocarditis (EMC) virus was studied by the use of light-sensitive virus grown in the presence of acridine orange (HRV 2) and neutral red (EMC). HeLa cells were protected against infection with HRV 2 by NH4Cl, monensin, and other compounds known to increase the pH of intracellular vesicles. Preincubation of the cells with the same compounds reduced the ability of the cells to bind [35S]methionine-labeled HRV 2, apparently due to inhibition of recycling of endocytosed receptors back to the cell surface. The cells were also protected against infection when HRV 2 was bound to cells on ice and the cells were then incubated at 37 degrees with the different compounds. This indicates that low pH is also necessary for some event in the entry process taking place after the virus is bound to the cells. In contrast, compounds which increase the pH in acidic intracellular compartments did not protect mouse L-cells against infection with EMC-virus, and the entry of the virus was inhibited by low pH in the medium. This inhibition was partly overcome by the presence of the ionophore monensin, which elevates the pH in endosomes and lysosomes. Possibly, EMC virus enters the cytosol from vesicles with neutral or slightly alkaline pH.

Animals↗
PubMed1984

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments.

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.

Ammonium Chloride↗
PubMed1984

Mechanism of entry into the cytosol of poliovirus type 1: requirement for low pH.

The effect of a number of drugs and culture conditions on the entry into cells of a strain of poliovirus 1 (Brunende) was tested. The cells were exposed in the dark to light-sensitive, neutral red-containing virus, in the presence of the drug to be tested. Then the cells were exposed to light, transferred to normal medium, and incubated overnight. Cytopathogenic effect was measured as inhibition of [3H]leucine incorporation. Compounds that dissipate proton gradients across membranes, like monensin, protonophores, and amines, and compounds that inhibit the acidification process, such as N,N'-dicyclohexylcarbodiimide (DCCD) and tributyltin, inhibited the entry of virus, but not virus binding. This was also the case with metabolic inhibitors that deplete cells for ATP. The same compounds also inhibited the cell-induced alteration of the virus particles. When cells with surface-bound virus were exposed to low pH, the virus entered efficiently, even in the presence of monensin and DCCD. The results indicate that acidification somehow facilitates the entry of the virus RNA into the cytosol and that under normal conditions the entry occurs from intracellular acidic vesicles.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1984

Effect of malignant transformation, retinoic acid, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) on the sensitivity of rodent cells to Pseudomonas toxin.

A number of mouse and rat cells and their virus-transformed counterparts were tested for sensitivity to Pseudomonas aeruginosa exotoxin A (PEA). In each case, the transformed cells were considerably less sensitive than were the nontransformed cells. In the presence of trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, or retinoic acid, the transformed cells became as sensitive as the nontransformed cells, whereas these drugs had little or no effect on the sensitivity to PEA of the nontransformed cells. Temperature-sensitive virus-transformed normal rabbit kidney cells were sensitized to PEA by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, when these cells were grown as the transformed phenotype, whereas the nontransformed phenotype could not be sensitized. The possibility is discussed that upon malignant transformation a process which is dependent upon calmodulin or protein kinase C strongly decreases the sensitivity of the cells to PEA.

ADP Ribose Transferases↗
PubMed1983

Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain.

Hybrid molecules were prepared from the A- and B-chains of the two toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic to Vero cells than were the parent toxins, native ricin and modeccin. A number of drugs (NH4Cl, monensin, trifluoperazine, verapamil, ionophore A23187) which protect cells against modeccin, but not against ricin, protected to some extent against the toxic hybrid, but less so than against native modeccin. The possibility is discussed that the modeccin A-chain of the hybrid may enter the cytosol by two routes, one which is highly efficient and identical to that used by native modeccin and another less efficient one which cannot be used by native modeccin.

Ammonium Chloride↗
PubMed1983

Dansylcadaverine eliminates calmodulin stimulation of phosphodiesterase.

Dansylcadaverine, which structurally resembles the calmodulin antagonists W-7 and W-5, prevented the calmodulin dependent stimulation of 3':5'-cyclic nucleotide phosphodiesterase in vitro. Dansylcadaverine and trifluoperazine sensitized cells to Pseudomonas aeruginosa exotoxin A in apparently the same way, exept that 40 times higher concentrations of dansylcadaverine than of trifluoperazine was required.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Isolation and characterization of viscumin, a toxic lectin from Viscum album L. (mistletoe).

A toxic protein, viscumin, was isolated from extracts of mistletoe by affinity chromatography on acid-treated Sepharose 4B. Viscumin was selectively bound to the column and could be eluted with lactose. It migrated in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponding to Mr = 60,000. In addition, two bands migrating corresponding to Mr = 29,000 and 32,000 were found. After treatment with 2-mercaptoethanol, only 2 bands (Mr = 29,000 and 34,000) were found. Apparently, viscumin consists of two chains which, in some of the molecules, are disulfide-linked. Protection experiments with antiserum against viscumin indicated that the major part of the cytotoxic activity in mistletoe extracts is due to viscumin. Gel filtration experiments on Sephacryl 200 indicated that, at low concentrations, viscumin occurs as a monomer and at higher concentrations as a dimer. Viscumin was found to inhibit protein synthesis in cell-free systems. When the two constituent peptide chains of viscumin were eluted from polyacrylamide gels and tested for ability to inhibit cell-free protein synthesis, this property was found to be associated with the fastest migrating chain, here denoted the A chain. The heavier chain was denoted the B chain. The A chain was found to inhibit protein synthesis by inactivating the ribosomes catalytically. Reconstitution experiments with isolated ribosomal subunits from untreated and A chain-treated ribosomes showed that the 60 S ribosomal subunit was selectively inactivated.

Animals↗
PubMed1982

Action of viscumin, a toxic lectin from mistletoe, on cells in culture.

A toxin from mistletoe, viscumin, inhibited the incorporation of leucine in cells more rapidly than the incorporation of uridine and thymidine, indicating that the toxins act by inhibiting cellular protein synthesis. The presence of galactose, lactose, and melibiose in the medium protected cells against viscumin. The sensitivity to viscumin of different cell lines differed considerably. The cytotoxic properties of viscumin were compared to those of the related toxins abrin, ricin, and modeccin. The profile of the sensitivity of a panel of cell lines to viscumin was dissimilar from those obtained with abrin and modeccin. Cell lines selected for resistance to modeccin and ricin were fully sensitive to viscumin. Ca2+ was required for viscumin to express its toxic effect. In contrast, the Ca2+ ionophore A23187 protected the cells against viscumin. Whereas the sensitivity of cells to viscumin did not vary much between pH 7 and 9, the cells were much less sensitive at pH 6. Also, cells treated with the two metabolic inhibitors, 2-deoxyglucose and NaN3, were insensitive to the toxins. Viscumin taken up by cells in the absence of Ca2+ or at pH 6 was able to intoxicate the cells when Ca2+ was added or when the pH was adjusted to neutrality. In contrast, cells exposed to viscumin in the presence of 2-deoxyglucose and NaN3, which inhibit endocytosis, were not intoxicated when the cells were treated with antiviscumin and then transferred to normal medium. The results indicate that endocytosis is involved in the entry of viscumin.

Animals↗
PubMed1982

Preparation and properties of chimeric toxins prepared from the constituent polypeptides of diphtheria toxin and ricin. Evidence for entry of ricin A-chain via the diphtheria toxin pathway.

A highly toxic conjugate of ricin A-chain and diphtheria toxin fragment B was prepared by disulfide exchange reaction. A similar conjugate between diphtheria toxin fragment A and ricin B-chain was nontoxic. Like native diphtheria toxin, the conjugate ricin A/diphtheria toxin B was much more toxic to Vero than to HeLa cells. Ricin was equally toxic to these cell lines. Lactose, which inhibits ricin binding, did not protect against the conjugate. Cells resistant to ricin, partly due to a reduced number of ricin-binding sites, were fully sensitive to the conjugate, indicating that the conjugate binds to diphtheria toxin receptors. The conjugate was fully toxic to two Vero cell mutants, resistant to diphtheria toxin because the elongation factor 2 could not be ADP-ribosylated by the diphtheria toxin A-fragment. Therefore, the inhibition of protein synthesis by the conjugate must be caused by the ricin A-chain. Ammonium chloride which prevents entry of diphtheria toxin, but not of ricin, also protected against the conjugate. Like diphtheria toxin, the conjugate was most toxic at low pH, whereas ricin is most active at pH above neutrality and inactive at low pH. The results indicate that the conjugate ricin A/diphtheria toxin B binds to diphtheria toxin receptors and inhibits cellular protein synthesis due to the action of ricin A-chain which appears to enter the cell by the diphtheria toxin pathway.

Ammonium Chloride↗