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Induction of apoptotic cell death by the mycotoxin gliotoxin and related toxins
562
Tenth World Congress
Study on fibrinolytic and fibrinogenolytic enzymes from the venom ofTrimeresurus mucrosquamatus. W. WANG, Y. XIONG, Y. ZZ-tANG,N. Lro, J. SONGand W.LI (Kunming Institute of Zoology, Academia Sinica, P.R. China). BY MEANSof DEAE-A 50, G-100, G-75, CM-CL 6B and FPLC columns, six fibrinolytic and fibrinogenolytic enzymes were purified to one band, respectively, on SDS-PAGE. All are glycoproteins and named for TMVF 1--6, with mol. wts 33,000, 32,000, 29,000 27,000, 26,000 and 24,300, and isoelectric points (p/) 9.3, 8.9, 8.1, 5.2, 4.6 and 4.2, respectively. They are all stable to acidic, alkaline and hot treatments, are activated by Ca 2+ and Mg 2+, and inhibited by Cu 2+, Fe 2+, Zn 2+ and Co 2÷. Components purified from peaks 1-5 of DEAE A-50 have proteolytic activities, those from peaks 12-14 display arginine esterase activities which are inhibited by PMSF but not by EDTA. This means they are serine proteinases. TMVF 3-5 have stronger hydrolytic activities on the thrombin substrate B2-Phe-VaI-Arg-PNA, the other demonstrates hydrolytic activities on fibrinolytic substrate CB2--GIy-Pro-Arg-PNA and this activity increases in the presence of plasma and serum. TMVF 1, 2 and 6 can hydrolyse the plasma and whole blood clots within 24 hr. TMVF 3 and 5 hydrolyse ~t-chain of fbrin mainly and then t-chain with a longer time. TMVF 1, 2 and 6 hydrolyse t-chain mainly and then ,,-chain in a longer time. Incubating TMVF 5 with fibrinogen significantly prolongs the thrombin time. Incubating TMVF 1-6 with fibrinogen for 30 min then adding thrombin reduces the content of soluble fibrin. The comparison of fibrin fragments was also carried out and is reported in another paper. Comparison o f detection of ricin in castor bean extracts by bioassays, immunoassays, and chemical procedures. R. W. WANr~4ACrmR JR, J. F. HEW~TSON, P. V. LEMLEY, M. A. POLl, R. E. DINTV~MAN, W. L. THOMPSONand D. R. FI~ANZ (Pathophysiology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-501 l, U.S.A.). RICIN is a 64,000 tool. wt protein from the seed of the castor bean plant. Castor meal, a by-product of castor oil production, was extracted with 0.02 N hydrochloric acid, acetone precipitated, and dissolved in saline. The resulting extract was analysed for ricin content by bioassay, immunoassay, and chemical procedures. Assays for detection (ppm sensitivity) were developed for ricin: mouse toxicity (0.4), Vero cell cytotoxicity (0.01), ELISA (0.002), HPLC (5.0), gel electrophoresis (20), and high performance capillary electrophoresis (CE) (25). By mouse bioassay, cell cytotoxicity assay, ELISA, HPLC, gel electrophoresis or CE, respectively, the ficin content of the extract (containing 20mg protein/ml) was 4.1, 4.9, !.3, 9.3, 3.3 and 2.9mg/ml. Both a goat anti-ricin polyclonal and a mouse anti-ricin A-chain monoclonal antibody neutralized the toxicity of the castor meal extract, which confirms that ricin is the toxic component of this extract. In conclusion, ricin could be detected in a crude extract of castor meal by /n vivo and in vitro bioassay, ELISA, or chemical procedures, but some difference was observed in quantitation by these procedures. This variation may be related to the presence of a protein matrix in the crude extract and/or the inability to resolve the ricin peak in the chemical procedures. Induction o f apoptotic cell death by the mycotoxin gliotoxin and related toxins. P. WAGING(John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra City 2601, Australia). GLIOTOXlN, a member of the epipolythiodioxopiperazine (ETP) class of secondary metabolities, shows potent immunosuppressive activity in vitro. It has been used successfully in a murine model to prevent tissue rejection and graft vs. host disease following pretreatment of tissue prior to transplantation. Gliotoxin appears to show selectivity towards mature cells of the immune system. Following treatment of cells with gliotoxin, typical morphological features of apoptotic cell death are apparent, as is the characteristic internucleosomal cleavage of DNA seen in this physiologically important form of cell death. We are currently studying the mechanism of induction of apoptosis by gliotoxin in a number of cell types in vitro. Gliotoxin causes apoptotic cell death in thymocytes, T-blasts and peritoneal macrophages. Apoptosis induced by gliotoxin in thymocytes cannot be blocked by protein synthesis inhibitors. This is in contrast to dexamethasone-induced cell death, which has led to the notion that apoptosis requires protein synthesis. Gliotoxin, in fact, inhibits protein synthesis and we have now shown that apoptosis can be induced by a number of protein synthesis inhibitors. Inhibition of protein synthesis as a trigger for apoptosis suggests the expression of a normal cellular protein responsible for inhibition of this form of cell death. We have also investigated the possible role of Ca 2+ in giiotoxin-induced cell death. Using 45Ca2+ we have measured uptake of calcium into treated cells and shown that gliotoxin does appear to induce influx of calcium into apoptotic cells. However, EGTA blocks this uptake at concentrations which do not affect DNA fragmentation, suggesting that extracellular calcium may not be important in apoptotic cell death. Identification o f tetramine as toxin causing food poisoning in Atlantic Canada following consumption o f whelks (Neptunea decemcostata). W. WATSON-WRIGHT,G. SIMS, C. SMYTH, M. GILLIS, M. MAHER, T. VANTROTTIER,D. SINCLAIR and M. GmOAN (Fisheries and Oceans Canada, Inspection Services Branch, Halifax, Nova Scotia, Canada). THREE recent incidents of toxin-like food poisoning in the Bay of Fundy area of Nova Scotia, Canada, which were associated with the consumption of boiled whelks, led to the discovery that the ten-ridged whelk (Neptunea
Tenth W o r d Congress
563
decemcostata) contains the salivary toxin tetramine (tetramethylammonium chloride; TMAC). Symptoms in all eases included visual problems (blurred and double vision), lightheadedness, 'depersonalization' or a feeling of 'being in the clouds' and weakness. In all three incidents, the onset of symptoms was within 30 rain following consumption and all but two of the 15 people affected recovered within a few hr. Those two who had consumed the largest quantity (approximately 15 whelks each) were nauseated for 48 hr in addition to suffering from the other symptoms. In the past, whelk-related poisonings in this area have traditionally been attributed to the presence of elevated levels of paralytic shellfish poison (PSP) in the digestive gland. In these incidents, mouse bioassay of digestive gland extracts prepared according to the A.O.A.C. procedure for paralytic shellfish poison confirmed the presence of only low levels of PSP ( < 40#g/100g tissue). However, bioassay of salivary gland extracts resulted in symptoms in the mice which were identical to those elicited by injection of TMAC and which included trembling, body flattening, fasciculations along the spine, excessive salivation (frothing at the mouth) and death within 1 to 2 rain. Chemical confirmation of the identity of TMAC was achieved through thin-layer chromatography followed by Dragendorff reagent spray. The estimated average concentration of TMAC in the salivary gland of this species was 9.4mg/100g shellfish as determined from a bioassay lethal dose curve. Although tetramine has previously been identified in related species (N. arthritica, N. antiqua, N. intersculpta) this represents the first report of the presence of tetramine in N. decemcostata. This discovery, combined with the perennial hazard of PSP in shellfish from the Bay of Fundy, has resulted in a permanent ban on the harvesting of whelks from this body of water. Bistratene A, an ascidian toxin, induces changes in the protein phosphorylation patterns o f human carcinoma cells. D. WATTERS(Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, 300 Herston Rd, Herston, Qld 4006, Australia). THE MECHANISMof action of a novel toxin has been investigated. Bistratene A is a macrocyclic polyether toxin isolated from the colonial ascidian Lissoclinum bistratum. It is toxic to tumour cells in vitro with an Icso value of 70 nM for T24 bladder carcinoma cells. We have previously shown that bistratene A causes the incomplete differentiation of HL-60 human leukemia cells at a concentration of 50riM, well below its 1¢50 value for these cells (300nM). The compound also causes growth inhibition of A549 human lung carcinoma cells at 10riM. These effects do not appear to be related to the enzyme protein kinase C, a key regulatory enzyme thought to be involved in the regulation of cellular growth and differentiation, since bistratene A is unable to activate the enzyme in vitro except at micromolar concentrations, and it does not inhibit the binding of phorbol dibutyrate to the enzyme. Two-dimensional gel electrophoretograms of phosphorylated proteins in HL-60 cells have been studied in control cells, and cells treated with bistratene A, phorbol ester and bryostatin. Bistratene A caused a unique pattern of phosphorylation distinct from that induced by the other differentiating agents. In particular, the phosphorylation and amount of a cytoplasmic protein of mol. wt 21,000 and p l 6.7 was rapidly increased by very low concentrations of bistratene. These results suggest that the compound interacts with some component of the signal transduction machinery and thus provides a unique tool with which to investigate cell differentiation mechanisms in tumour cells. Inhibition o f the proteolytic activity o f haemorrhagin-e from Crotalus atrox venom by purified antihaemorrhagins o f homologous serum. S. WEtSSSENBVaG,~'2 M. OVADIA~ and E. KOCHVAl (1Department of Zoology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel; ~ p e n University of Israel, P.O.B. 39328, Tel-Aviv 61392, Israel). ANTIHAEMORRHAGICproteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of haemorrhagic toxin-e from Crotalus atrox venom and of several proteolytic enzymes--trypsin, collagenase and thermolysin. Purified antihaemorrhagic proteins inhibited the proteolytic activity of haemorrhagln-e when tested on gelatin type I, the proteolytic activity of trypsin when tested on photofilm (gelatin) and the proteolytic activity of whole venom when tested on azocollagen and photofilm. The antihaemorrhaglns failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-fl-naphthyl ester (APNE), the activity of collagenase on N-furylacryloyl-Leu-GlyPro-Ala (FALGPA) or on azocollagen, and the activity of thermolysin on N-furylacryloyl-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihaemorrhaglns from snake blood serum are proteolytic inhibitors that underwent some specialization towards the neutralization of haemorrhaglc toxins through their antiproteolytic activity. Spiderbite: an overview. J. WHITE (State Toxinology Services, Adelaide Children's Hospital, North Adelaide, SA 5006, Australia). STATISTICS on spiderbite are less extensive than those for snakebite, but available evidence suggests that while mortality from spiderbite is low compared to snakebite, total numbers of cases, and eases with morbidity
Tenth World Congress
Study on fibrinolytic and fibrinogenolytic enzymes from the venom ofTrimeresurus mucrosquamatus. W. WANG, Y. XIONG, Y. ZZ-tANG,N. Lro, J. SONGand W.LI (Kunming Institute of Zoology, Academia Sinica, P.R. China). BY MEANSof DEAE-A 50, G-100, G-75, CM-CL 6B and FPLC columns, six fibrinolytic and fibrinogenolytic enzymes were purified to one band, respectively, on SDS-PAGE. All are glycoproteins and named for TMVF 1--6, with mol. wts 33,000, 32,000, 29,000 27,000, 26,000 and 24,300, and isoelectric points (p/) 9.3, 8.9, 8.1, 5.2, 4.6 and 4.2, respectively. They are all stable to acidic, alkaline and hot treatments, are activated by Ca 2+ and Mg 2+, and inhibited by Cu 2+, Fe 2+, Zn 2+ and Co 2÷. Components purified from peaks 1-5 of DEAE A-50 have proteolytic activities, those from peaks 12-14 display arginine esterase activities which are inhibited by PMSF but not by EDTA. This means they are serine proteinases. TMVF 3-5 have stronger hydrolytic activities on the thrombin substrate B2-Phe-VaI-Arg-PNA, the other demonstrates hydrolytic activities on fibrinolytic substrate CB2--GIy-Pro-Arg-PNA and this activity increases in the presence of plasma and serum. TMVF 1, 2 and 6 can hydrolyse the plasma and whole blood clots within 24 hr. TMVF 3 and 5 hydrolyse ~t-chain of fbrin mainly and then t-chain with a longer time. TMVF 1, 2 and 6 hydrolyse t-chain mainly and then ,,-chain in a longer time. Incubating TMVF 5 with fibrinogen significantly prolongs the thrombin time. Incubating TMVF 1-6 with fibrinogen for 30 min then adding thrombin reduces the content of soluble fibrin. The comparison of fibrin fragments was also carried out and is reported in another paper. Comparison o f detection of ricin in castor bean extracts by bioassays, immunoassays, and chemical procedures. R. W. WANr~4ACrmR JR, J. F. HEW~TSON, P. V. LEMLEY, M. A. POLl, R. E. DINTV~MAN, W. L. THOMPSONand D. R. FI~ANZ (Pathophysiology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-501 l, U.S.A.). RICIN is a 64,000 tool. wt protein from the seed of the castor bean plant. Castor meal, a by-product of castor oil production, was extracted with 0.02 N hydrochloric acid, acetone precipitated, and dissolved in saline. The resulting extract was analysed for ricin content by bioassay, immunoassay, and chemical procedures. Assays for detection (ppm sensitivity) were developed for ricin: mouse toxicity (0.4), Vero cell cytotoxicity (0.01), ELISA (0.002), HPLC (5.0), gel electrophoresis (20), and high performance capillary electrophoresis (CE) (25). By mouse bioassay, cell cytotoxicity assay, ELISA, HPLC, gel electrophoresis or CE, respectively, the ficin content of the extract (containing 20mg protein/ml) was 4.1, 4.9, !.3, 9.3, 3.3 and 2.9mg/ml. Both a goat anti-ricin polyclonal and a mouse anti-ricin A-chain monoclonal antibody neutralized the toxicity of the castor meal extract, which confirms that ricin is the toxic component of this extract. In conclusion, ricin could be detected in a crude extract of castor meal by /n vivo and in vitro bioassay, ELISA, or chemical procedures, but some difference was observed in quantitation by these procedures. This variation may be related to the presence of a protein matrix in the crude extract and/or the inability to resolve the ricin peak in the chemical procedures. Induction o f apoptotic cell death by the mycotoxin gliotoxin and related toxins. P. WAGING(John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra City 2601, Australia). GLIOTOXlN, a member of the epipolythiodioxopiperazine (ETP) class of secondary metabolities, shows potent immunosuppressive activity in vitro. It has been used successfully in a murine model to prevent tissue rejection and graft vs. host disease following pretreatment of tissue prior to transplantation. Gliotoxin appears to show selectivity towards mature cells of the immune system. Following treatment of cells with gliotoxin, typical morphological features of apoptotic cell death are apparent, as is the characteristic internucleosomal cleavage of DNA seen in this physiologically important form of cell death. We are currently studying the mechanism of induction of apoptosis by gliotoxin in a number of cell types in vitro. Gliotoxin causes apoptotic cell death in thymocytes, T-blasts and peritoneal macrophages. Apoptosis induced by gliotoxin in thymocytes cannot be blocked by protein synthesis inhibitors. This is in contrast to dexamethasone-induced cell death, which has led to the notion that apoptosis requires protein synthesis. Gliotoxin, in fact, inhibits protein synthesis and we have now shown that apoptosis can be induced by a number of protein synthesis inhibitors. Inhibition of protein synthesis as a trigger for apoptosis suggests the expression of a normal cellular protein responsible for inhibition of this form of cell death. We have also investigated the possible role of Ca 2+ in giiotoxin-induced cell death. Using 45Ca2+ we have measured uptake of calcium into treated cells and shown that gliotoxin does appear to induce influx of calcium into apoptotic cells. However, EGTA blocks this uptake at concentrations which do not affect DNA fragmentation, suggesting that extracellular calcium may not be important in apoptotic cell death. Identification o f tetramine as toxin causing food poisoning in Atlantic Canada following consumption o f whelks (Neptunea decemcostata). W. WATSON-WRIGHT,G. SIMS, C. SMYTH, M. GILLIS, M. MAHER, T. VANTROTTIER,D. SINCLAIR and M. GmOAN (Fisheries and Oceans Canada, Inspection Services Branch, Halifax, Nova Scotia, Canada). THREE recent incidents of toxin-like food poisoning in the Bay of Fundy area of Nova Scotia, Canada, which were associated with the consumption of boiled whelks, led to the discovery that the ten-ridged whelk (Neptunea
Tenth W o r d Congress
563
decemcostata) contains the salivary toxin tetramine (tetramethylammonium chloride; TMAC). Symptoms in all eases included visual problems (blurred and double vision), lightheadedness, 'depersonalization' or a feeling of 'being in the clouds' and weakness. In all three incidents, the onset of symptoms was within 30 rain following consumption and all but two of the 15 people affected recovered within a few hr. Those two who had consumed the largest quantity (approximately 15 whelks each) were nauseated for 48 hr in addition to suffering from the other symptoms. In the past, whelk-related poisonings in this area have traditionally been attributed to the presence of elevated levels of paralytic shellfish poison (PSP) in the digestive gland. In these incidents, mouse bioassay of digestive gland extracts prepared according to the A.O.A.C. procedure for paralytic shellfish poison confirmed the presence of only low levels of PSP ( < 40#g/100g tissue). However, bioassay of salivary gland extracts resulted in symptoms in the mice which were identical to those elicited by injection of TMAC and which included trembling, body flattening, fasciculations along the spine, excessive salivation (frothing at the mouth) and death within 1 to 2 rain. Chemical confirmation of the identity of TMAC was achieved through thin-layer chromatography followed by Dragendorff reagent spray. The estimated average concentration of TMAC in the salivary gland of this species was 9.4mg/100g shellfish as determined from a bioassay lethal dose curve. Although tetramine has previously been identified in related species (N. arthritica, N. antiqua, N. intersculpta) this represents the first report of the presence of tetramine in N. decemcostata. This discovery, combined with the perennial hazard of PSP in shellfish from the Bay of Fundy, has resulted in a permanent ban on the harvesting of whelks from this body of water. Bistratene A, an ascidian toxin, induces changes in the protein phosphorylation patterns o f human carcinoma cells. D. WATTERS(Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, 300 Herston Rd, Herston, Qld 4006, Australia). THE MECHANISMof action of a novel toxin has been investigated. Bistratene A is a macrocyclic polyether toxin isolated from the colonial ascidian Lissoclinum bistratum. It is toxic to tumour cells in vitro with an Icso value of 70 nM for T24 bladder carcinoma cells. We have previously shown that bistratene A causes the incomplete differentiation of HL-60 human leukemia cells at a concentration of 50riM, well below its 1¢50 value for these cells (300nM). The compound also causes growth inhibition of A549 human lung carcinoma cells at 10riM. These effects do not appear to be related to the enzyme protein kinase C, a key regulatory enzyme thought to be involved in the regulation of cellular growth and differentiation, since bistratene A is unable to activate the enzyme in vitro except at micromolar concentrations, and it does not inhibit the binding of phorbol dibutyrate to the enzyme. Two-dimensional gel electrophoretograms of phosphorylated proteins in HL-60 cells have been studied in control cells, and cells treated with bistratene A, phorbol ester and bryostatin. Bistratene A caused a unique pattern of phosphorylation distinct from that induced by the other differentiating agents. In particular, the phosphorylation and amount of a cytoplasmic protein of mol. wt 21,000 and p l 6.7 was rapidly increased by very low concentrations of bistratene. These results suggest that the compound interacts with some component of the signal transduction machinery and thus provides a unique tool with which to investigate cell differentiation mechanisms in tumour cells. Inhibition o f the proteolytic activity o f haemorrhagin-e from Crotalus atrox venom by purified antihaemorrhagins o f homologous serum. S. WEtSSSENBVaG,~'2 M. OVADIA~ and E. KOCHVAl (1Department of Zoology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel; ~ p e n University of Israel, P.O.B. 39328, Tel-Aviv 61392, Israel). ANTIHAEMORRHAGICproteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of haemorrhagic toxin-e from Crotalus atrox venom and of several proteolytic enzymes--trypsin, collagenase and thermolysin. Purified antihaemorrhagic proteins inhibited the proteolytic activity of haemorrhagln-e when tested on gelatin type I, the proteolytic activity of trypsin when tested on photofilm (gelatin) and the proteolytic activity of whole venom when tested on azocollagen and photofilm. The antihaemorrhaglns failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-fl-naphthyl ester (APNE), the activity of collagenase on N-furylacryloyl-Leu-GlyPro-Ala (FALGPA) or on azocollagen, and the activity of thermolysin on N-furylacryloyl-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihaemorrhaglns from snake blood serum are proteolytic inhibitors that underwent some specialization towards the neutralization of haemorrhaglc toxins through their antiproteolytic activity. Spiderbite: an overview. J. WHITE (State Toxinology Services, Adelaide Children's Hospital, North Adelaide, SA 5006, Australia). STATISTICS on spiderbite are less extensive than those for snakebite, but available evidence suggests that while mortality from spiderbite is low compared to snakebite, total numbers of cases, and eases with morbidity