Enterococcus faecalis plasmid pAD1-encoded Fst toxin affects membrane permeability and alters cellular responses to lantibiotics

Fst is a peptide toxin encoded by the par toxin-antitoxin stability determinant of Enterococcus faecalis plasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.     

Interaction of T-2 toxin and murine lymphocytes and the demonstration of a threshold effect on macromolecular synthesis

Although T-2 toxin intoxications have been described as radiomimetic, we find that T-2 toxin does not preferentially affect multiplying cells. Among the targets of T-2 toxin toxicity, DNA, RNA and protein synthesis inhibition are analysed. All three types of macromolecular syntheses are affected by a threshold dose of T-2 toxin which corresponds to the interaction of approx. 1 X 10(5) T-2 toxin molecules with the same number of T-2 toxin receptors (Gyongyossy-Issa, M.I.C. and Kachatourians, G.G. (1984) Biochim. Biophys. Acta 803, 197-202). Since toxic effects occur faster at higher toxin concentrations than at lower levels, the time-toxic effect relationship may be defined by a constant. Based on these observations, we hypothesize that complete receptor-occupation is the critical first step in the course of T-2 toxin toxicity events.     

Cholera toxin B-subunit protects mammalian cells from ricin and abrin toxicity

The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.     

Action of oligomycin on cultured mammalian cells. Permeabilization to translation inhibitors

Summary Oligomycin, an inhibitor of ATP synthesis, has been used as a model to study the effects of ATP depletion on macromolecular synthesis and modification of membrane permeability. Protein synthesis is totally blocked by the antibiotic, whereas RNA and DNA synthesis are less inhibited. Different concentrations of monovalent and divalent cations do not revert the inhibition of protein synthesis. Measurement of cellular ATP and ⁸⁶Rb⁺ content indicate that the blockade of translation depends on the ATP content. A significant decrease in cellular ATP does not lead to the reduction of monovalent ions in the cell, although hyperpolarization of the cell membrane does take place. An increased membrane permeability to some inhibitors develops when the cells are hyperpolarized by oligomycin.     

Interaction of pseudomonas exoproducts with phagocytic cells

Polymorphonuclear leukocytes play the major role in host defense against infections with Pseudomonas aeruginosa; however, mononuclear cells also may contribute to defense against pulmonary infections with P. aeruginosa. Therefore, we examined the effects of three extracellular products of P. aeruginosa, exotoxin A, alkaline protease, and elastase, on the function of phagocytic cells. Phagocytosis or killing, protein synthesis, and membrane integrity were used as assays of cellular function. Pseudomonas toxin readily inhibited protein synthesis in mouse peritoneal macrophages; in contrast, proteolytic enzymes did not alter protein synthesis, but transiently decreased the sensitivity of macrophages to toxin. High levels of toxin reduced protein synthesis in human peripheral polymorphonuclear leukocytes but did not alter the ability of these cells to kill P. aeruginosa. Elastase and alkaline protease did not cause release of marker enzymes and did not directly inhibit the bactericidal activity of polymorphonuclear leukocytes; killing was reduced due to inactivation of complement components. In conclusion, these potential virulence products do not modify phagocyte function directly and thus do not directly interfere with host response in pseudomonas infections.     

The relative effects of different types of growth factors on DNA replication, mitosis, and cellular enlargement

It was recently demonstrated that growth in cell size can be dissociated from DNA synthesis and mitosis. 3T3 cells starved to quiescence in low serum concentration can be stimulated to undergo DNA synthesis and one cell division without growing in size (unbalanced growth) (42-44). We report here that in cells stimulated to undergo unbalanced growth, the cell nucleus undergoes balanced growth, i.e., nearly doubles in size prior to mitosis. The reduced ability to grow in cell size under unbalanced growth conditions is thus mainly ascribable to the cytoplasm. Furthermore, the extent to which cells grow in size prior to mitosis is dependent on the serum concentration in the tissue culture medium (44). This data suggests that some macromolecular factor or factors in serum are required for growth in cell size prior to mitosis. We report in this study that epidermal growth factor (EGF) alone exerts a small but significant stimulatory influence on DNA synthesis and mitosis but does not affect cellular enlargement. In contrast, insulin added at supraphysiological concentrations does not stimulate quiescent cells to enter S phase but instead stimulates growth in cell size in the small fraction of dividing cells. Furthermore, cells stimulated to proliferate by EGF could be induced to undergo balanced growth when insulin was added concomitantly. Finally, platelet-derived growth factor (PDGF) stimulates quiescent sparse 3T3 cells to undergo DNA synthesis and mitosis. PDGF also exerts a limited but significant effect on cellular enlargement. However, PDGF alone could not induce a complete balanced growth, i.e., a doubling in cell size prior to mitosis.     

Serotonin-induced deoxyribonucleic acid synthesis in vascular smooth muscle cells involves a novel, pertussis toxin-sensitive pathway

Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.     

Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts

In cultured human lung fibroblasts treated with Clostridium difficile cytotoxin, the latency before appearance of the cytopathogenic effect was dose-related with a minimum of 45 min. At 37 degrees C, the toxin was accessible on all cells to inactivation with trypsin or neutralization with antitoxin during the first tenth of the latency. At 0 degrees C, the toxin was accessible considerably longer. The cytopathogenic effect was reversibly prevented by the lysosomotropic agents chloroquine and ammonium chloride, which had to be added within one-fifth of the latency to protect all cells. In the presence of chloroquine, but not of ammonium chloride, the time period during which the toxin remained amenable to neutralization with antitoxin was prolonged. The protective effect of ammonium chloride was not influenced by dropping the extracellular pH to 4.5, but that of chloroquine was abolished. The expression of the intoxication was not affected by inhibitors of the DNA, RNA or protein synthesis. Inhibitors of the energy metabolism prevented the cytopathogenic effect when added before the last phase of the latency. The results suggest that expression of the cytopathogenic effect requires internalization of the toxin, and that metabolic energy but no macromolecular synthesis is needed for the action of the toxin after this internalization.     

Effects of Oleate Starvation in a Fatty Acid Auxotroph of Escherichia coli K-12

The effects of oleate starvation on an oleate auxotroph of Escherichia coli K-12 were investigated. Following removal of oleate from the mutant growing in a minimal glycerol-peptone medium, the cells stopped making deoxyribonucleic acid, ribonucleic acid, protein, and phospholipids; they began to die exponentially and finally lysed. During oleate starvation in minimal medium minus peptone, inhibition of macromolecular syntheses and death occurred; however, lysis did not follow. When growth ceased, no further dying was observed. It is shown that none of the early effects (inhibition of macromolecular syntheses and death) can be due to leakiness of the cells, induction of a prophage or a colicin, or lack of energy sources. The cause of inhibition of macromolecular syntheses remained unknown. Since the rate of death was the same as the generation time under different conditions, it appears that death is due to the defective synthesis of some cellular structure (quite possibly, cytoplasmic membrane) during phospholipid deficiency. Lysis was found to require protein synthesis; electron microscopy revealed a peculiar type of "lysis from within"; i.e., the shape of the cells did not change but fragmentation of the inner layer of the cell envelope occurred. The murein was found to be unaltered. Most likely, lysis was a consequence of the cell's attempt to synthesize cytoplasmic membrane with altered phospholipid composition or during phospholipid deficiency. Several membrane functions (respiration, adenosine triphosphate formation, permeability) existing before oleate removal were not lost during starvation. Therefore, general damage to the membrane did not occur, and it could be that most, if not all, described effects were due to defective de novo membrane synthesis.     

Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells

The purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L-cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP-elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP-elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP-stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid-insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose-response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP-stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP-elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.     

Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts

In cultured human lung fibroblasts treated with Clostridium difficile cytotoxin, the latency before appearance of the cytopathogenic effect was dose-related with a minimum of 45 min. At 37°C, the toxin was accessible on all cells to inactivation with trypsin or neutralization with antitoxin during the first tenth of the latency. At 0°C, the toxin was accessible considerably longer. The cytopathogenic effect was reversibly prevented by the lysosomotropic agents chloroquine and ammonium chloride, which had to be added within one-fifth of the latency to protect all cells. In the presence of chloroquine, but not of ammonium chloride, the time period during which the toxin remained amenable to neutralization with antitoxin was prolonged. The protective effect of ammonium chloride was not influenced by dropping the extracellular pH to 4.5, but that of chloroquine was abolished. The expression of the intoxication was not affected by inhibitors of the DNA, RNA or protein synthesis. Inhibitors of the energy metabolism prevented the cytopathogenic effect when added before the last phase of the latency. The results suggest that expression of the cytopathogenic effect requires internalization of the toxin, and that metabolic energy but no macromolecular synthesis is needed for the action of the toxin after this internalization.     

Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 μg/ml. ¹²⁵I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH₄Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.     

Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.     

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.     

Increases in cyclic AMP potentiate competence formation in BALB/c-3T3 cells

The ability of platelet-derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c-3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of mediuum supplemented with platelet-poor plasma. The enhancement of serum-induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma-controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet-derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c-3T3 cells were increased 2- to 3-fold after the addition of cholera toxin. The addition of cholera toxin plus 30 m?M isobutylmethylxanthine caused an even greater (7- to 8-fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8-bromocyclic AMP also caused a concentration-dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet-derived growth factor.     

Lack of influence of mitochondrial genetical information on the multiplication of Herpes simplex virus in HeLa cells

Mitochondrial DNA synthesis in HeLa cells is inhibited by 0.2 μg ethidium bromide/ml whereas nuclear DNA synthesis is essentially unimpaired under the same conditions. The action of ehtidium bromide on mitochondrial DNA appears to be completed within 18 hours of exposure to the drug. Total cellular macromolecular synthesis under ethidium bromide is initially decreased and at later times slightly stimulated. Ethidium bromide pretreatment of HeLa cells did not significantly affect the multiplication of Herpes simplex virus as compared with that in control cells.     

Histidyl-Transfer Ribonucleic Acid Synthetase in Positive Control of the Histidine Operon in Salmonella typhimurium

Autolysin-like enzymes appear to be responsible for cell separation in Agmenellum quadruplicatum. Mutants that are impaired in cell separation and grow as chains exhibit reduced cell lytic activity. Lysozyme, extracted autolysin, and antibiotics that affect peptidoglycan synthesis phenotypically suppress chain formation. Various aspects of the regulation of the cell separation process were also examined. Studies involving antibiotic inhibitors of macromolecular synthesis and general growth inhibitors provided no evidence for the active regulation of the cell separation process during the latter portion of the division cycle. Evidence was obtained, however, for the partial restriction of peptidogly-can hydrolysis by unknown secondary modifications. The thin electron-dense layer of peptidoglycan along the sides of cells was much more resistant to hydrolysis by egg-white lysozyme than was the septum between daughter cells. The middle portion of the septum was more sensitive than was the layer immediately adjacent to the cytoplasmic membrane. Under conditions that would not osmotically stabilize spheroplasts, lysozyme facilitates rapid cell separation in chain-forming mutants with little leakage of cellular protein or loss of viability.     

A comparative study of ricin and diphtheria toxin-antibody-conjugate kinetics on protein synthesis inactivation

Kinetic data on toxin and antibody-toxin-conjugate inactivation of protein synthesis have been used to assess the variables which affect the transport of these toxins into the cytosol compartment. First-order inactivation rate constants of protein synthesis (ki) are compared under conditions of known receptor occupancy. The effect of inclusion of toxin B chains, both homologous and heterologous, in antibody-toxin conjugates is observed, and factors which affect toxin lag periods are studied. The results show that the inclusion of B chains in conjugates increases ki values 3-10-fold, but only if the B chain is homologous with the A chain. In spite of the augmentation of antibody-toxin-conjugate ki values by homologous toxin B chain, these ki values are only 1/20 those observed with unmodified toxins on sensitive cells. A further difference noted between toxins and antibody-toxin conjugates is the presence of a dose-dependent lag when toxins, but not antibody-toxin conjugates, effect sensitive cell types. This lag period for ricin can be shortened by alkalinizing the cell medium. The kinetic data can be fit by assuming a processing step interposed between the binding of ricin to surface receptors and the interaction of the A chain with ribosomes which is first-order in toxin concentration and pH-dependent. The time constant of this event is reflected in the dose-dependent lag period. It is proposed that antibody-toxin conjugates do not participate in this processing event and therefore fail to achieve the high entry levels exhibited by unmodified toxins.