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Results for “DIPHTHERIA TOXIN”

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At least 19 recordsLinked to original sources
PubMed1991

Influenza virus enhancement of membrane leakiness induced by staphylococcal alpha toxin, diphtheria toxin and streptolysin S.

Release of alpha-amino[14C]isobutyric acid from ferret Mpf cells was promoted by staphylococcal alpha toxin, diphtheria toxin and streptolysin S. This release was enhanced to a significant extent if the cells had been previously infected with influenza virus strain A/Puerto Rico/8/34 (PR8, H1N1), although infection with virus alone did not increase the release of radiolabel as compared with that from untreated cells; inactivated virus had a similar enhancing action. The mechanism of enhancement is unclear but it occurs between 0.5 and 2h post-inoculation and viral membrane/endosome membrane fusion is essential. Endotoxin had no effect on membrane permeability, either alone or with PR8. The relevance of these in vitro observations to the previously observed enhancement of toxin lethality by influenza virus in vivo is discussed.

Aminoisobutyric Acids↗
PubMed1996

Comparison of the conformation, hydrophobicity, and model membrane interactions of diphtheria toxin to those of formaldehyde-treated toxin (diphtheria toxoid): formaldehyde stabilization of the native conformation inhibits changes that allow membrane insertion.

Toxoids are inactivated protein toxins that are used in vaccines. The behavior of diphtheria toxin reacted with formaldehyde (diphtheria toxoid) was compared to that of diphtheria toxin in order to understand the nature of the changes that occur in toxoids upon protein reaction with formaldehyde. Despite the intramolecular cross-links in the toxoid, the conformations of the toxoid and the toxin were very similar in both the native and low pH-induced membrane-penetrating states as judged by fluorescence and hydrophobicity properties. However, the toxoid underwent thermal-, low-pH-, and guanidinium chloride-induced conformational changes only at more extreme conditions than needed to induce such changes in the toxin. This implies that formaldehyde modification stabilizes the native conformation relative to several conformations that involve different degrees of unfolding. The stabilization to conformational changes induced by low pH is particularly interesting because low pH induces partial unfolding of the toxin to a molten globule-like state. It was found that the toxoid only gained the ability to interact with model membrane vesicles at a lower pH than the toxin. Because low-pH-induced unfolding and membrane interaction are critical steps in the entry of diphtheria toxin into cells, the resistance of the toxoid to these changes may be linked to its lack of toxicity. The implications of these results for the construction of toxoids are discussed.

Binding Sites↗
PubMed1999

The effects of inhibitors upon pore formation by diphtheria toxin and diphtheria toxin T domain.

The formation of pores by membrane-inserted diphtheria toxin is closely linked to the translocation of its catalytic chain across membranes. In this report a number of aromatic polyanionic molecules were identified that inhibit toxin-induced leakage of molecules from model membrane vesicles. One inhibitor, Cibacron blue, totally blocked pore formation. Aniline blue and Fast Green decreased the size of the molecule released by a given concentration of toxin. Amaranth appeared to reduce the maximal amount of leakage, without greatly affecting the size of the molecule released at a given toxin concentration. Finally, Ponceau S and Cibacron brilliant red appeared to exhibit a mixture of these various types of inhibition. The inhibitors neither prevented the conformational transition of the toxin to form a hydrophobic state at low pH, nor (with the exception of Cibacron Brilliant Red) appeared to strongly inhibit toxin binding to model membranes. Additional experiments showed release of trapped materials from model membranes by isolated T domain of the toxin was similar to that by whole toxin. The effects of inhibitors on T domain induced release was also similar to that they have on whole toxin. Therefore, it is likely that the inhibition of pore formation by whole toxin involves inhibitor interaction with the T domain. The inhibitors identified in this study may be helpful for development of agents that interfere with toxin action in vivo.

Aniline Compounds↗
PubMed1998

[The protective effect of the nerve growth factor in exposing a nerve tissue culture to diphtheria toxin].

Diphtheria toxin (1.10(-1)-1.10(-6) Lf/ml) was found to inhibit neurite extension in chick embryo dorsal root ganglia in vitro. If the nerve growth factor (60 ng/ml) was added with toxin in culture media the diphtheria toxin effect was decreased and the neurite outgrowth was compared with control. Protective effect of nerve growth factor by influence of diphtheria toxin may be used in new principles of diphtheria treatment.

Animals↗
PubMed1995

[The protective effect of the nerve growth factor on sensory neurons in organotypic culture under the action of diphtheria toxin].

Diphtheria toxin (1.10(-1) - 1.10(-6) Lf/ml) was found to inhibit neurite extension in chick embryo dorsal root ganglia in vitro. If the nerve growth factor (60 ng/ml) was added with the toxin to culture media, the effect of diphtheria toxin was seen to decrease and the neurite outgrowth was compared with control. Protective effect of nerve growth factor estimated by the influence of diphtheria toxin may be used for new principles of diphtheria treatment.

Animals↗
PubMed1984

Thermal stability of different forms of diphtheria toxin.

Diphtheria toxin and its enzymatically active A fragment have been examined by differential scanning calorimetry. The thermal stability was measured for different forms of these molecules, including tryptically nicked and intact, nucleotide-bound and free, cysteine alkylated, and cysteine oxidized and reduced. Three ranges of denaturation temperature have been observed among the different forms of diphtheria toxin studied, and there is a correlation of the thermal stability of these different forms with their biological activity. At the concentrations used for measurement, all forms of the 62,000-Da diphtheria toxin are irreversibly denatured by heating to 100 degrees C, while free A chain is reversibly denatured. In vivo and in vitro activities of the samples were measured before and after heating, and all were found to retain significant degrees of activity after heating.

Diphtheria Toxin↗
PubMed1992

Ion channel and membrane translocation of diphtheria toxin.

Diphtheria toxin is the best studied member of a family of bacterial protein toxins which act inside cells. To reach their cytoplasmic targets, these toxins, which include tetanus and botulinum neurotoxins and anthrax toxin, have to cross the hydrophobic membrane barrier. All of them have been shown to form ion channels across planar lipid bilayer and, in the case of diphtheria toxin, also in the plasma membrane of cells. A relation between the ion channel and the process of membrane translocation has been suggested and two different models have been put forward to account for these phenomena. The two models are discussed on the basis of the available experimental evidence and in terms of the focal points of difference, amenable to further experimental investigations.

Animals↗
PubMed1973

Biological activity of heated diphtheria toxin.

Diphtheria toxin splits into two fragments when heated at 100 C for 10 min in a phosphate buffer. The separated fragments have molecular weights of 24,000 and 39,000, respectively. These molecular weights are similar to those of the A and B fragments found in diphtheria toxin preparations after thiol reduction. Since the separation of toxin into fragments is not complete, it is likely that only nicked toxin molecules having a cleaved peptide bond are split by heating. When toxin is suspended in phosphate buffer at pH 6.4, the B-like fragment precipitates, but at pH 7.8 it does not. Heated toxin is unable to intoxicate sensitive cells or cause a necrodermal response in animals. Fragment A produced by heating is active in inhibiting cell-free protein synthesis. It is able to intoxicate both HeLa and L cells when the uptake of the fragment is facilitated by addition of polyornithine to the cultures. Fragment B produced by heating is involved with binding to the cell surface. It is able to delay the action of toxin on KB cell cultures preincubated with fragment B.

Animals↗
PubMed1978

Demyelination of Sternarchus electrocyte fibers by injection of diphtheria toxin.

Diphtheria toxin was injected into the electric organ of the gymnotid fish, Sternarchus albifrons. After 10 days, there was extensive demeylination of electrocyte fibers in the area of injection. Electron microscopy showed that paranodal loops of myelin do not separately cleanly from the axon, and remnants of the myelin loops may persist after demyelination of the internodes is nearly complete. The dense cytoplasmic undercoating of the nodal axolemma may disappear before the paranodal junctions are completely gone. Observations of demyelination of internodes between the elaborate, inexcitable nodes suggest that the presence of myelin may not be necessary for the maintenance of structural differentiation of this region of the axolemma. Use of diphtheria toxin to demyelinate Sternarchus electrocytes may provide a useful system for experimental neuropathological studies.

Animals↗
PubMed1985

1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin.

Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed.

Adenosine Diphosphate↗
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↗
PubMed1995

Protective effect of cell-permeable ceramide analogs against modeccin, ricin, Pseudomonas toxin, and diphtheria toxin.

We investigated the effects of various ceramide (Cer) analogs and related sphingolipids on the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in various cell lines. The most pronounced protective effect by C6Cer, a short-chain cell-permeable Cer analog, was observed in modeccin cytotoxicity in Vero, BER-40, and MDCK cells, whereas the cytotoxicity of diphtheria toxin was not affected by any of the ceramide analogs tested. C6Cer did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells. C2Cer and C8Cer also protected against modeccin cytotoxicity, albeit less effectively than C6Cer. However, related sphingolipids including sphingosine, sphingomyelin, lactosylceramide, C18Cer (the naturally occurring ceramide), and dihydro C6Cer had no effect. A correlation was found between the ability of ceramides to inhibit bulk protein secretion and the inhibition of modeccin cytotoxicity by ceramides. Among Cer analogs tested, C6Cer, the most potent inhibitor of modeccin cytotoxicity, strongly inhibited bulk protein secretion in Vero, BER-40, and MDCK cells. PtK1 cells, which were not protected by ceramides against toxins, were resistant to ceramide-induced inhibition of bulk protein secretion. These results confirm that Cer may modulate the intracellular transport of proteins through the Golgi complex. Such Cer-sensitive processes may be involved in the intoxication of cells by plant and bacterial toxins, especially modeccin.

Animals↗
PubMed2000

Requirement for prolonged action in the cytosol for optimal protein synthesis inhibition by diphtheria toxin.

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.

Animals↗
PubMed1982

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.

Cell Division↗
PubMed1992

Expression of functional diphtheria toxin receptors on highly toxin-sensitive mouse cells that specifically bind radioiodinated toxin.

Diphtheria toxin (DT), a bacterial protein exotoxin, inactivates mammalian cell elongation factor 2 after toxin internalization by receptor-mediated endocytosis. To isolate the DT receptor, we cotransfected DT-resistant wild-type mouse L-M cells with a cDNA library constructed from RNA of highly toxin-sensitive monkey Vero cells and with a neomycin-resistance gene. Stably transfected G418-resistant L-M colonies were screened for DT sensitivity in a replica plate assay. After screening of 8000 colonies, one DT-sensitive (DTS) colony was isolated. The purified DTS mouse cells are highly toxin-sensitive; they are at least 1000-fold more sensitive than wild-type L-M cells and only approximately 10-fold less sensitive than Vero cells. Incubation of the DTS mouse cells with CRM 197, a nontoxic form of DT that competitively inhibits the binding of native DT to the toxin receptor, protected them from DT-mediated toxicity. More important, these DTS mouse cells express receptors on their cell surface that bind radioiodinated DT in a specific fashion, a property hitherto readily demonstrable only with highly toxin-sensitive cells of monkey origin. Furthermore, HA6DT, a DT fragment comprising the Mr 6000 carboxyl-terminal receptor-binding domain, inhibited the binding of radioiodinated toxin to these DTS mouse cells to the same extent as unlabeled DT. With these DTS mouse cells as a source of monkey cDNA, it should be possible to clone the gene encoding the DT receptor.

Animals↗
PubMed1982

X-ray grade crystals of diphtheria toxin.

Diphtheria toxin, complexed with the endogenous dinucleotide ApUp, has been crystallized under novel conditions involving high concentrations of both polyethylene glycol and salt. Among four crystal forms obtained, one was found suitable for high resolution structural analysis by x-ray diffraction. This form is triclinic (space group P1) with 2 molecules per unit cell; unit cell parameters are a = 70.8 A, b = 70.7 A, c = 65.3 A, alpha = 95.2 degrees, beta = 91.3 degrees, gamma = 99.7 degrees.

Crystallization↗
PubMed1984

A functional role for cysteine disulfides in the transmembrane transport of diphtheria toxin.

Diphtheria toxin was modified at one or both of its cysteine disulfide bridges by iodoacetamide, methylmethanethiosulfonate, and atomic mercury. The products of these reactions were characterized and tested for toxicity in vitro and in vivo. All were toxic in vitro, but had lost almost all cytotoxic activity toward HeLa cells. It was possible to show from in vivo protection experiments that modification of the cysteine disulfide in the B-chain interfered with cell surface binding, while modification of the cysteine disulfide linking the A and B domains inhibits a step subsequent to binding in the intoxication process. The latter finding supports a functional role for this interdomain cysteine disulfide in the membrane transport process.

Adenosine Diphosphate Ribose↗
PubMed1990

Morphometric evidence from C-synapses for phased Nissl body response in alpha-motoneurones retrogradely intoxicated with diphtheria toxin.

Diphtheria toxin (DTX) kills cells by inactivating ribosomal translocation and when used to retrogradely intoxicate cat intercostal motoneurones produces marked morphological alterations in Nissl bodies, including those specifically sited postsynaptic to C-type axon terminals. Here, qualitative examinations of 'intoxicated' postsynaptic Nissl bodies reveal a progressive structural alteration marked by rER dilatation, rER lamellae fragmentation but retention of both the highly ordered multilamellate organization and ribosomal attachment until final stages of Nissl body dissolution. Morphometric results identified 3 broad phases to the postintoxication response which differed in the degree of rER cisternal dilation, and the numerical and spatial relationships between rER-lamellae, rER-bound ribosomes and rER-associated polyribosomes. These phases reflect the known molecular basis of diphtheritic toxicity and contrast with the fast developing Nissl body reaction associated with the neurotoxin ricin which also invokes ribosomal dysfunction and has been used to mimic certain features of motor neurone disease. The cytopathology of DTX and ricin are compared in the Discussion.

Animals↗