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Use of oligonucleotide probes in the cloning of epsilon toxin from Clostridium perfringens type B.
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9th World Congress
in loss of its activity to block vWF as well as fibrinogen binding to human platelets. Intravenous infusion of trigramin into hamster resulted in a significant prolongation of the mesentery bleeding time. In conclusion, our data suggest the biological activity of trigramin may depend upon the presence of RGD and upon the secondary structure of this molecule. REFERENCE HUANG, T. F., HOLT, J. C., LUKASIEWICZ,H. and NIEWIAROWSKI,S. (1987) J. bioL Chem. 262, 16157.
Effects of congeners of macrocyclic trichothecene mycotoxins on the murine immune system. B. J. HUGHES,1 B. B. JARVlS,2 G. C. HSIEH1 and R. P. SHARMA~ (XCenter for Environmental Toxicology, Utah State University, Logan, UT 84322-5600, and 2Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, U.S.A.). MACROCYCLIC trichothecenes are a class of mycotoxins recently isolated and evaluated for their anti-leukemic properties. We studied 11 of these mycotoxins: roritoxin B, myrotoxin B, roridin A, verrucarin A, 16-OH verrucarin A, verrucarin J, baccharinoid B12, rofidin D, roridin E, baccharinoid B4 and baccharinoid B5 for their immunotoxicity in CD-1 mice. An equitoxic dose was prepared in 1% DMSO in saline and administered i.p. at half the LDso. Organ weights, WBCs, RBCs, differentials, blastogenesis of splenic lymphocytes in response to concanavalin A (Con A), lipopolysaccharide (LPS), phytohemagglutinin (PHA) and pokeweed mitogen (PWM), and mixed lymphocyte reaction (MLR) were studied on day 4 after administration. Organ weights showed significant differences in the baccharinoids with a decrease in spleen weight in B 12 and an increased liver weight in B4 and B5 treated animals. Differentials of WBCs were unremarkable while myrotoxin B, verrucarin J, roridin A and roridin E had statistically different WBC counts. Myrotoxin B showed a decrease in RBCs. The baccharinoids BI2 and B4 showed significant increases in the MLR. Roritoxin B and baccharinoid B5 increased Con A stimulation. Roridin A and baccharinoid BI2 increased LPS stimulation with baccharinoid B5 decreasing the LPS response. Stimulation with PHA was significantly increased by roridin A and baccharinoid B5. Responses to PWM remained unaltered. Anti-SRBC antibodies were significantly decreased in the roridin D and E and baccharinoid B5-treated animals. Supported in part by PHS-ES 07097 and by PHS-CA 25967.
Use of oligonucleotide probes in the cloning of epsilon toxin from Clostridium perfringens type B. SOPHIE E. C. HUNTER and RICHARD W. TITnALL (Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K.). CLOSTRIDIUMPERFRINGENStype B produces an inactive prototoxin, epsilon prototoxin, which is converted to active toxin when digested with trypsin. It is one of the major toxins produced by the type B strain. The toxin is associated with a number of diseases in animals, including lamb dysentery and enterotoxaemia in sheep, goats and foals (HAUSCmLD, 1971). We decided to clone the epsilon toxin gene, with a view to producing genetically manipulated toxoids which may be used as vaccines. We also want to investigate, by genetic means, the activation of the prototoxin. As a prerequisite of cloning, we required a way of identifying the clone. Two possible routes were available to us: to use antibodies to identify the gene product, or to use a DNA probe to identify the gene. We chose the latter route, since the amino acid sequence from the N-terminus of epsilon prototoxin had been previously determined (BHowN and HABEEB, 1977). We have re-sequenced the N-terminus of purified epsilon prototoxin and used the data to design a number of oligonucleotide probes. The probes are being used to explore the possibility of cloning the epsilon toxin gene. REFERENCES BnOWN, A. S. and HABEEB,A. F. S. A. (1977) Biochem. biophys. Res. Commun. 78, 889. HAUSCHILD, A. H. W. (1971) In: Microbial Toxins, Vol. 11A, p. 159 (KADIS, MONTIE and AJL, Eds). New York: Academic Press.
Studies of the ionic channel properties of anatoxin-a(s). EDWARD G. HYDE1 and WAYNE W. CARMICHAEL2 (1Biomedical Sciences Ph.D. Program and 2Department of Biological Sciences, Wright State University, Dayton, OH 45435, U.S.A.). ANATOXlN-a(s) [antx-a(s)], a potent anticholinesterase isolated from the cyanobacterium Anabaena tips-aquae NRC-525-17, has been shown to have no intrinsic agonist activity at muscarinic receptors but does sensitize the denervated guinea pig ileum to muscarinic agonists. Potassium chloride depolarization of frog rectus abdominis
9th World Congress
in loss of its activity to block vWF as well as fibrinogen binding to human platelets. Intravenous infusion of trigramin into hamster resulted in a significant prolongation of the mesentery bleeding time. In conclusion, our data suggest the biological activity of trigramin may depend upon the presence of RGD and upon the secondary structure of this molecule. REFERENCE HUANG, T. F., HOLT, J. C., LUKASIEWICZ,H. and NIEWIAROWSKI,S. (1987) J. bioL Chem. 262, 16157.
Effects of congeners of macrocyclic trichothecene mycotoxins on the murine immune system. B. J. HUGHES,1 B. B. JARVlS,2 G. C. HSIEH1 and R. P. SHARMA~ (XCenter for Environmental Toxicology, Utah State University, Logan, UT 84322-5600, and 2Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, U.S.A.). MACROCYCLIC trichothecenes are a class of mycotoxins recently isolated and evaluated for their anti-leukemic properties. We studied 11 of these mycotoxins: roritoxin B, myrotoxin B, roridin A, verrucarin A, 16-OH verrucarin A, verrucarin J, baccharinoid B12, rofidin D, roridin E, baccharinoid B4 and baccharinoid B5 for their immunotoxicity in CD-1 mice. An equitoxic dose was prepared in 1% DMSO in saline and administered i.p. at half the LDso. Organ weights, WBCs, RBCs, differentials, blastogenesis of splenic lymphocytes in response to concanavalin A (Con A), lipopolysaccharide (LPS), phytohemagglutinin (PHA) and pokeweed mitogen (PWM), and mixed lymphocyte reaction (MLR) were studied on day 4 after administration. Organ weights showed significant differences in the baccharinoids with a decrease in spleen weight in B 12 and an increased liver weight in B4 and B5 treated animals. Differentials of WBCs were unremarkable while myrotoxin B, verrucarin J, roridin A and roridin E had statistically different WBC counts. Myrotoxin B showed a decrease in RBCs. The baccharinoids BI2 and B4 showed significant increases in the MLR. Roritoxin B and baccharinoid B5 increased Con A stimulation. Roridin A and baccharinoid BI2 increased LPS stimulation with baccharinoid B5 decreasing the LPS response. Stimulation with PHA was significantly increased by roridin A and baccharinoid B5. Responses to PWM remained unaltered. Anti-SRBC antibodies were significantly decreased in the roridin D and E and baccharinoid B5-treated animals. Supported in part by PHS-ES 07097 and by PHS-CA 25967.
Use of oligonucleotide probes in the cloning of epsilon toxin from Clostridium perfringens type B. SOPHIE E. C. HUNTER and RICHARD W. TITnALL (Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K.). CLOSTRIDIUMPERFRINGENStype B produces an inactive prototoxin, epsilon prototoxin, which is converted to active toxin when digested with trypsin. It is one of the major toxins produced by the type B strain. The toxin is associated with a number of diseases in animals, including lamb dysentery and enterotoxaemia in sheep, goats and foals (HAUSCmLD, 1971). We decided to clone the epsilon toxin gene, with a view to producing genetically manipulated toxoids which may be used as vaccines. We also want to investigate, by genetic means, the activation of the prototoxin. As a prerequisite of cloning, we required a way of identifying the clone. Two possible routes were available to us: to use antibodies to identify the gene product, or to use a DNA probe to identify the gene. We chose the latter route, since the amino acid sequence from the N-terminus of epsilon prototoxin had been previously determined (BHowN and HABEEB, 1977). We have re-sequenced the N-terminus of purified epsilon prototoxin and used the data to design a number of oligonucleotide probes. The probes are being used to explore the possibility of cloning the epsilon toxin gene. REFERENCES BnOWN, A. S. and HABEEB,A. F. S. A. (1977) Biochem. biophys. Res. Commun. 78, 889. HAUSCHILD, A. H. W. (1971) In: Microbial Toxins, Vol. 11A, p. 159 (KADIS, MONTIE and AJL, Eds). New York: Academic Press.
Studies of the ionic channel properties of anatoxin-a(s). EDWARD G. HYDE1 and WAYNE W. CARMICHAEL2 (1Biomedical Sciences Ph.D. Program and 2Department of Biological Sciences, Wright State University, Dayton, OH 45435, U.S.A.). ANATOXlN-a(s) [antx-a(s)], a potent anticholinesterase isolated from the cyanobacterium Anabaena tips-aquae NRC-525-17, has been shown to have no intrinsic agonist activity at muscarinic receptors but does sensitize the denervated guinea pig ileum to muscarinic agonists. Potassium chloride depolarization of frog rectus abdominis