Effect of ionizing radiation on Fc-receptors of mouse peritoneal mononuclear cells

The influence of different gamma-radiation doses on the number of mouse peritoneal mononuclears bearing Fc-receptors was studied. At doses of 7 and 9.5 Gy, phase changes were observed in the number of cells bearing Fc-receptors: a short-term rise registered 24 h to 2 days after irradiation was followed by depression. In addition, the total number of peritoneal mononuclears decreased. At a dose of 100 Gy, the number of cells with Fc-receptors was reduced as early as 1 h after irradiation, and the pronounced cell lysis occurred.     

Effects of ultraviolet irradiation on structural and functional condition of T- and B-lymphocytes of human blood

The influence of ultraviolet (UV) radiation on structural and functional condition of membranes T- and B-lymphocytes of human blood is investigated. It is shown, that intergral flow of UV-light (240-390 nm) in doses 151-1359 J/m2 leads to the increase of the expression of some HLA-antigen (DR1, DR2, DR5, DR7) and Fc-receptors on membranes of B-lymphocytes. The level of HLA-antigen was increased by 20-500% from the initial state after exposure to different doses. The growth of the expression of Fc-receprots was registered in 80% of the donors in the range from 20 up to 540%. The minimum dose of UV-light (151 J/m2) caused the maximum effect. The reduction of the level of Fc-receptors by 10-90% relatively to the native condition was registered for T-cells in 80% of the donors. Groups of the donors were revealed, T-lymphocytes of which differed by the sensitivity to UV-radiation. The effect of "strengthening--weakening", resulting to the reduction of the initially high parameters and to increase in the initially low ones was found.     

Action of different toxins from the scorpion Androctonus australis on a locust nerve-muscle preparation

The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.     

Reductive chain separation of botulinum A toxin--a prerequisite to its inhibitory action on exocytosis in chromaffin cells

Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release of catecholamines from permeabilized chromaffin cells [(1989) FEBS Lett. 242, 245-248; (1989) J. Neurochem., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond. The actions of both neurotoxins on exocytosis were presently compared using streptolysin O-permeabilized bovine adrenal chromaffin cells. Botulinum A toxin inhibited Ca2+-stimulated catecholamine release from these cells. Addition of dithiothreitol lowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five. This indicates that the interchain S-S bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.     

Effects of four trichothecene mycotoxins on activation marker expression and cell proliferation of human lymphocytes in culture

Four trichothecene mycotoxins – the type A trichothecenes T2-toxin and diacetoxyscirpenol and the type B trichothecenes nivalenol and deoxynivalenol – were studied. The effects of these mycotoxins on the expression of the sequentially expressed activation markers CD69, CD25, and CD71 and on proliferation of human lymphocytes were studied in culture with a duration of up to 72 h.All the examined toxins affected activation marker expression in a similar way. After 6 h, the CD69 expression was lower in exposed cultures compared to controls. After 24 and 48 h of exposure, an increased frequency of cells expressing CD69 was found in exposed cultures, indicating a delay in downregulation of CD69 expression. Stimulation of CD25 expression was observed for doses below the IC₅₀ value, while suppression was found for higher doses. The pattern was different from that detected for CD69 expression, in that an increased expression of CD25 never occurred after exposure to the highest concentration of the toxin, and in that no stimulatory effects were found after 48 h of exposure, indicating that the response was inhibited and not delayed. The effects of toxin exposure on CD71 expression were in many respects similar to the effects on CD25 expression.We conclude that the trichothecene mycotoxins investigated in this study inhibited the cell cycle in a similar way and exert their main antiproliferative action rather early in the cell cycle, before or in conjunction with CD25 expression.     

Interaction of blood lymphocyte Fc receptors with IgG during in vitro cultivation

Bovine blood lymphocytes taken from normal cows and those suffering from chronic lymphocytic leukemia were cultured in complete medium 199 with 10% of heat-inactivated fetal bovine serum. After a 48 hour culturing an enhanced quantity of the Fc-receptor bound fluoresceinated immunoglobulin G (IgG) was established. When lymphocyte fractions enriched with T- or B-cells were cultured, the binding capacity of Fc-receptors for IgG (48 hours after culture) increased in both the cell populations. A study of the kinetics of interactions of Fc-receptors with IgG showed that the increased number of Fc-receptors after culturing was followed by an enhanced affinity of Fc-receptors towards IgG. The affinity of Fc-receptors of blood lymphocytes of cows with chronic lymphocytic leukemia was lower than that of normal lymphocytes.     

Cholera toxin inhibits the increase in cytoplasmic free calcium induced via the CD2 pathway of human T-lymphocyte activation

We investigated the action of cholera toxin on the intracellular ionized calcium [Ca2+]i increase induced by anti-CD2 and anti-CD3 monoclonal antibodies in the leukemic human T-cell line Jurkat. Cholera toxin inhibits in a dose-dependent manner these two pathways of human T-lymphocyte activation but with different half maximal inhibition doses (75 ng/ml for CD3, 30 ng/ml for CD2). This effect cannot be accounted for only by the increase in cAMP induced by cholera toxin because forskolin, which raises cellular cyclic adenosine monophosphate (cAMP) to the same levels, induced only a small inhibition of the [Ca2+]i increase in similar conditions. Cholera toxin induced a decrease in the surface expression of the CD3 molecule, suggesting a down-regulation of the CD3 molecules. On the other hand, the expression of CD2 remained unchanged. Cell surface disappearance of the CD3 molecule cannot account for all the inhibitory effects of cholera toxin because CD2 molecule expression was not affected (no modifications in the half maximal binding of anti-CD2 monoclonal antibodies). All together, these results suggest that cholera toxin acts on substrates, possibly G proteins, that could regulate the [Ca2+]i increase induced by anti-CD2 and anti-CD3 mAbs in Jurkat cells. In addition, the present study demonstrated that the rise in cellular cAMP partially inhibits the [Ca2+]i increase induced by anti-CD2 and anti-CD3 mAbs.     

Competitive relations between tetanus anatoxin and toxin]

Experiments were conducted on albino mice; it was shown that preliminary injection of tetanus toxoid enhanced the animal resistance to tetanus toxin, this being expressed in increase in LD50. The effect increased the higher doses of the toxoid and their fractional injection. By using protagon and crude mitochondrial fraction isolated from the brain as a receptor of tetanus toxin in the nervous tissue there were established competitive relations for the receptor between the tetanus toxoid and the toxin. The results of investigations confirmed the authors' earlier statement that the molecule of the tetanus toxin contained different functional groups responsible for the toxin binding with the receptor in the nervous tissue, for the pathogenic action of the toxin and for the binding of the toxin with antitoxin.     

Comparative evaluation of the neutrophilokine-inducing activity of Yersinia pestis EV and Escherichia coli lipopolysaccharides

As shown in this study, neutrophilokine-inducing capacity of Y. pestis EV lipopolysaccharide (LPS) was not inferior to, and in secondary immune response even exceeded, that of E. coli LPS. Neutrophilokines synthesized under the action of the former preparation produced greater influence on the inhibition of macrophage migration from the focus of infection, the phagocytic activity of these cells (in secondary immune response) and the labilization of the lysosomic membranes of macrophages than neutrophilokines induced by E. coli LPS. Only in primary immune response the digestive capacity of macrophages was more actively stimulated by neutrophilokines induced by E. coli LPS. Both preparations did not induce the secretion of neutrophilokines regulating the expression of Fc-receptors on the surface of macrophages.     

Studies on the intoxication pathway of tetanus toxin in the rat pheochromocytoma (PC12) cell line. Binding, internalization, and inhibition of acetylcholine release

Tetanus toxin was found to be a potent inhibitor of neurosecretion in the rat pheochromocytoma cell line PC12, a system in which biochemical and functional studies could be performed in parallel. Incubation of the cells with 10 nM tetanus toxin (3 h) led to an inhibition of acetylcholine release by 75-80% when evoked by 200 microM veratridine, 1 mM carbachol, or 2 mM Ba2+. The main characteristics of the inhibition process are: 1) the toxin is very potent, with threshold doses of 10 pM; 2) the action of toxin is blocked at low temperature (0 degrees C) and by antitoxin; 3) the effects are dose- and time-dependent; 4) a concentration-dependent lag phase precedes the onset of the inhibitory effects. Thus the PC12 cultures are a valid system for studies on the underlying molecular process in tetanus action. This system was exploited by the use of long term incubation studies to examine the processes responsible for the lag phase. When cells were incubated with 0.1 nM 125I-tetanus toxin, cell-associated toxin reached a plateau of 16 fmol of toxin/mg of protein, yet the toxic effects did not appear until 12 h. Further, PC12 cells were found to rapidly internalize tetanus toxin, with a half-life of 1-2 min, once it was bound to the surface of the cells. Thus, the lag phase results from steps that occur in the intracellular compartment after internalization. An important discovery was that the differentiation state of the PC12 cells was a critical factor in determining sensitivity to tetanus toxin. Cells that were cultured with nerve growth factor for 8-12 days were very sensitive to toxin. In contrast, acetylcholine release from nondifferentiated, autodifferentiated, or dexamethasone-treated cultures was insensitive to tetanus toxin. Since differential expression of high affinity tetanus toxin receptors cannot explain these results, it is concluded that PC12 cells are capable of expressing different forms of excitation-secretion coupling mechanisms. Tetanus toxin should prove a valuable probe to further distinguish these processes.     

Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.     

Evaluation of hypoxic state in experimental animals induced by Yersinia pestis antigens

Some characteristics of purine metabolism in experimental animals (white mice, clawed jirds and guinea pigs), injected intraperitoneally with Y. pestis "murine" toxin and capsular antigen (Fraction 1), were studied. Under the influence of sublethal doses of these antigens increased levels of guanine and xanthine in blood were noted. Changes in the content of xanthine oxidase in cells were insignificant. In white mice and clawed jirds the activity of succinate dehydrogenase decreased under the action of "murine" toxin and increased after the injection of Fraction 1.     

Properties and use of botulinum toxin and other microbial neurotoxins in medicine.

Crystalline botulinum toxin type A was licensed in December 1989 by the Food and Drug Administration for treatment of certain spasmodic muscle disorders following 10 or more years of experimental treatment on human volunteers. Botulinum toxin exerts its action on a muscle indirectly by blocking the release of the neurotransmitter acetylcholine at the nerve ending, resulting in reduced muscle activity or paralysis. The injection of only nanogram quantities (1 ng = 30 mouse 50% lethal doses [U]) of the toxin into a spastic muscle is required to bring about the desired muscle control. The type A toxin produced in anaerobic culture and purified in crystalline form has a specific toxicity in mice of 3 x 10(7) U/mg. The crystalline toxin is a high-molecular-weight protein of 900,000 Mr and is composed of two molecules of neurotoxin (ca. 150,000 Mr) noncovalently bound to nontoxic proteins that play an important role in the stability of the toxic unit and its effective toxicity. Because the toxin is administered by injection directly into neuromuscular tissue, the methods of culturing and purification are vital. Its chemical, physical, and biological properties as applied to its use in medicine are described. Dilution and drying of the toxin for dispensing causes some detoxification, and the mouse assay is the only means of evaluation for human treatment. Other microbial neurotoxins may have uses in medicine; these include serotypes of botulinum toxins and tetanus toxin. Certain neurotoxins produced by dinoflagellates, including saxitoxin and tetrodotoxin, cause muscle paralysis through their effect on the action potential at the voltage-gated sodium channel. Saxitoxin used with anaesthetics lengthens the effect of the anaesthetic and may enhance the effectiveness of other medical drugs. Combining toxins with drugs could increase their effectiveness in treatment of human disease.     

A functional domain on the alpha-latrotoxin molecule, distinct from the binding site, involved in catecholamine secretion from PC12 cells: identification with monoclonal antibodies

Seven monoclonal antibodies (mAbs) have been produced against alpha-latrotoxin (alpha-Latx), the toxin component of black widow spider venom that stimulates release of neurotransmitters from PC12 cells. These mAbs were characterized by an enzyme-linked immunosorbent assay and by neutralization analysis of the secretagogue properties of the toxin. The production of a panel of mAbs, possibly directed against different epitopes of alpha-Latx, provides a useful set of reagents to dissect the molecular regions of the toxin having different functions and to describe steps of its mode of action in responsive cells. Attention was focused on one of these mAbs (4C4.1), which inhibits in a dose-dependent fashion both toxin-stimulated and crude venom stimulated dopamine release from PC12 cells, prevents toxin-induced 45Ca2+ accumulation in PC12, alters toxin-dependent phosphoinositide breakdown, and prevents toxin-induced channel formation in artificial lipid bilayers. Since, within certain experimental conditions, mAb 4C4.1 is able to recognize the toxin bound to cells, we conclude that its effects were not a consequence of a direct interference with binding. On the basis of kinetic analysis of mAb interference on toxin action, expressed as accumulation of inositol phosphates and transmitter secretion, we suggest that the described effects result primarily from the blockade of an event immediately successive to binding and central for the full expression of toxin action. The availability of mAb 4C4.1 now makes possible the molecular characterization of the toxin moiety responsible for such an event.     

Novel yeast killer toxins provoke S-phase arrest and DNA damage checkpoint activation

Certain strains of Pichia acaciae and Wingea robertsiae (synonym Debaryomyces robertsiae) harbour extranuclear genetic elements that confer a killer phenotype to their host. Such killer plasmids (pPac1-2 of P. acaciae and pWR1A of W. robertsiae) were sequenced and compared with the zymocin encoding pGKL1 of Kluyveromyces lactis. Both new elements were found to be closely related to each other, but they are only partly similar to pGKL1. As for the latter, they encode functions mediating binding of the toxin to the target cell's chitin and a hydrophobic region potentially involved in uptake of a toxin subunit by target cells. Consistently, mutations affecting the target cell's major chitin synthase (Chs3) protect it from toxin action. Heterologous intracellular expression of respective open reading frames identified cell cycle-arresting toxin subunits deviating structurally from the likewise imported gamma-subunit of the K. lactis zymocin. Accordingly, toxicity of both P. acaciae and Wingea toxins was shown to be independent of RNA polymerase II Elongator, which is indispensable for zymocin action. Thus, P. acaciae and Wingea toxins differ in their mode of action from the G1-arresting zymocin. Fluorescence-activated cell sorting analysis and determination of budding indices have proved that such novel toxins mediate cell cycle arrest post-G1 during the S phase. Concomitantly, the DNA damage checkpoint kinase Rad53 is phosphorylated. As a mutant carrying the checkpoint-deficient allele rad53-11 displays toxin hypersensitivity, damage checkpoint activation apparently contributes to coping with toxin stress, rather than being functionally implemented in toxin action.     

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

Effect of dimethylnitrosamine on enzyme induction in rat liver

1. The effects of various doses of dimethylnitrosamine on the hydrocortisone induction of tryptophan pyrrolase were studied. A single LD(50) dose of dimethylnitrosamine inhibits the synthesis of the enzyme if given within the first 5hr. of the induction. A quarter of this dose also inhibits the synthesis of the enzyme, but, in addition, retards the rate of decay of the enzyme. 2. A single small dose of dimethylnitrosamine significantly inhibits the hydrocortisone induction of tryptophan pyrrolase for at least 14 days without causing widespread damage to liver substructure. 3. The inhibition by dimethylnitrosamine of induced tryptophan pyrrolase synthesis is probably independent of any action of the toxin on the synthesis of cofactors necessary for full expression of the enzyme's activity. Dimethylnitrosamine appears to act directly on the synthesis of the enzyme protein. 4. The synthesis of that form of the enzyme which is most sensitive to hydrocortisone is apparently also most susceptible to the action of dimethylnitrosamine. 5. It is suggested that the inhibition of protein synthesis by dimethylnitrosamine may be a result of methylation of messenger RNA, which then is unable to code effectively for amino acid polymerization.     

Expression of the surface membrane receptors of neutrophils in myeloproliferative diseases

For 147 patients with myeloproliferative diseases, a study was made of the expression of Fc-receptors to immunoglobulins IgC, IgA, and that of FcH-receptor and receptors to C3-components of the complement in peripheral blood neutrophils. The data obtained show the lower level of neutrophils having membrane receptors in patients with acute myeloblastic leukemia and chronic granulocytic leukemia at the stage of blast transformation. A decrease in expression of membrane receptors of neutrophils was shown in patients with chronic granulocytic leukemia and benign subleukemic myelosis. The finding of a higher level of neutrophils having surface receptors revealed in patients with real polycythemia is close to the data obtained in the study of expression of membrane receptors in patients with chronic myeloproliferative diseases and healthy persons.     

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity.

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1-4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat- and mouse-derived cell lines were very insensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line-specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.     

Yeast, beef and pork extracts counteract Clostridium difficile toxin A enterotoxicity

Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo . In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.