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Preparation and characterization of toxin-toxin conjugates as immunogens
52 6
Report and Abstracts
rest of the fusion protein using endoproteinase factor X . The cardiotoxin thus obtained showed cytotoxic effects on cultured Hehr cells. Preparation and characterization of toxin-toxin conjugates as immroeogens . P . V. LeMt~r, P. AMANATIDPS, J . Ruvo and T . Hootcaa (Tozinology Division, U .S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, U .S .A .). Tt~ are several challenges to achieving a good immunological response to biological toxins . Low mol. wt toxins will not evoke an immunological response unless conjugated to a carrier . Protein toxins are frequently detoxified with formaldehyde so that the immune response is not to the native protein . Achieving protective immunity in mice was a logical prelude to screening for toxin-neutralizing monoclonal antibodies . We were interested in multiple toxin immunity in mice and in toxin-neutralizing monoclonal antibodies . We investigated ricin as an immunogenic carrier for some low mol . wt (mw) haptens. Saxitoxin (mw = 301), palytoxin (mw = 2680), oonotoxin Gl (FW=1437 .6) and staphylococcal enterotoxin B (mw=43,111) were conjugated to ricin to evaluate the toxin-toxin conjugate . Conjugates were prepared by formalin activation (conjugation through free amines) . While in vitro toxicity could be reduced by three logs, compared to native toxin, in vivo toxicity was not predicted . Good immune responses to ricin (carrier) were always achieved but hapten titers varied. The problem with carrier-hapten stability in vivo is being addressed. The views of the author do not purport to reflect the positions of the Department of the Army or the Department of Defense. The experiments conducted therein were performed according to the principles set forth in the current edition of the Guide jor the Care and Use ojLaboratory Animals, Institute of Laboratory Animal Resources, National Research Council . A study on the cause of death dire to waglerin-1, a toxin from Trimeresurus wagleri . C. Y . LEe,' W . W . LtN' and L . A . SsurFtZ (' Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan; and ZU.S . Army Medical Research Institute of Infectious Disease, Fort Detrick, Frederick, MD 21702-5011, U .S .A.) . W~or .extx-1, a lethal toxin isolated from the venom of Tümeresurus wagleri, consists of 22 amino acid residues with a proline-rich sequence . In the present study, we investigated the cause of death and the mode of action of this toxin . In anaesthetized mice, i .v. administration of waglerin-I at 0.5 mg/kg produced respiratory failure within 5 min, and the blood pressure could be maintained by artificial respiration applied immediately after respiratory arrest. In anaesthetized rats, however, no toxic effects on respiration and blood pressure were observed up to l0 mg/kg (i.v .). Waglerin-I at 10 pg/ml reversibly blocked the indirect twitch of the mouse phrenic nerve-hemidiaphragm preparation, but had no effect on the direct twitch. In accordance with the in vivo experiments, waglerin-1 at concentrations up to 100 pg/ml did not block the indirect twitch of the rat diaphragm . In the chick biventer cervicis muscle, the amplitude of indirect twitch was decreased to about 15%,then gradually recovered to 80% of the pre-toxin level, by 30pg/ml toxin, and it was completely blocked by 1001rg/ml toxin at 10 min. The toxin (30 pg/ml) blocked the contracture induced by 30 pg/ml ACh, but not that induced by high KCI (30 mM) . It is wncluded that respiratory failure resulting from neuromuscular block is the primary cause of death due to waglerin-1 in mice and that rats are rather resistant to waglerin-1 . Thus, waglerin-I is a novel type neurotoxin in snake venoms . The views of the author do not purport to reflect the positions of the Department of the Army or the Department of Defense. The experiments conducted therein were performed aceording to the principles set forth in the current edition of the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council. Bothrojaracin, a potent and speck thrombin inhibitor from Bothrops jararaca venom . R. Ztxont.t,'~ M . JANDAOT-PERRUS, 3 M .-C. Gtnt.uN' and C. Horn' (' Unité des Venins, Institut Pasteur, Paris, France ; ZDepartamento do Bioquimica, Universidade Federal do Rio de Janeiro, Brazil; and'Hémostase et Thrombose, Faculté Xavier Bichat, Université Paris VII, Paris, France). A Ttntouumtx specific inhibitor, named bothrojaracin, has been identified in the venom of Bothrops jararaca and purified to homogeneity . It is an acidic protein of mol . wt 27,000 and is composed of two disulfide-linked subunits of 13,500 and 15,000 . It has some structural similarities with botrocetin, a component characterised in the same venom and which binds to von Willebrand factor and induces platelet agglutination . Bothrojaracin forms an equimolecular non~ovalent complex with human a-thrombin ; however, it does not affect the catalytic site of thrombin, since amidolytic activity, measured with a chromogenic substrate S-2238, remained unchanged in the
Report and Abstracts
rest of the fusion protein using endoproteinase factor X . The cardiotoxin thus obtained showed cytotoxic effects on cultured Hehr cells. Preparation and characterization of toxin-toxin conjugates as immroeogens . P . V. LeMt~r, P. AMANATIDPS, J . Ruvo and T . Hootcaa (Tozinology Division, U .S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, U .S .A .). Tt~ are several challenges to achieving a good immunological response to biological toxins . Low mol. wt toxins will not evoke an immunological response unless conjugated to a carrier . Protein toxins are frequently detoxified with formaldehyde so that the immune response is not to the native protein . Achieving protective immunity in mice was a logical prelude to screening for toxin-neutralizing monoclonal antibodies . We were interested in multiple toxin immunity in mice and in toxin-neutralizing monoclonal antibodies . We investigated ricin as an immunogenic carrier for some low mol . wt (mw) haptens. Saxitoxin (mw = 301), palytoxin (mw = 2680), oonotoxin Gl (FW=1437 .6) and staphylococcal enterotoxin B (mw=43,111) were conjugated to ricin to evaluate the toxin-toxin conjugate . Conjugates were prepared by formalin activation (conjugation through free amines) . While in vitro toxicity could be reduced by three logs, compared to native toxin, in vivo toxicity was not predicted . Good immune responses to ricin (carrier) were always achieved but hapten titers varied. The problem with carrier-hapten stability in vivo is being addressed. The views of the author do not purport to reflect the positions of the Department of the Army or the Department of Defense. The experiments conducted therein were performed according to the principles set forth in the current edition of the Guide jor the Care and Use ojLaboratory Animals, Institute of Laboratory Animal Resources, National Research Council . A study on the cause of death dire to waglerin-1, a toxin from Trimeresurus wagleri . C. Y . LEe,' W . W . LtN' and L . A . SsurFtZ (' Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan; and ZU.S . Army Medical Research Institute of Infectious Disease, Fort Detrick, Frederick, MD 21702-5011, U .S .A.) . W~or .extx-1, a lethal toxin isolated from the venom of Tümeresurus wagleri, consists of 22 amino acid residues with a proline-rich sequence . In the present study, we investigated the cause of death and the mode of action of this toxin . In anaesthetized mice, i .v. administration of waglerin-I at 0.5 mg/kg produced respiratory failure within 5 min, and the blood pressure could be maintained by artificial respiration applied immediately after respiratory arrest. In anaesthetized rats, however, no toxic effects on respiration and blood pressure were observed up to l0 mg/kg (i.v .). Waglerin-I at 10 pg/ml reversibly blocked the indirect twitch of the mouse phrenic nerve-hemidiaphragm preparation, but had no effect on the direct twitch. In accordance with the in vivo experiments, waglerin-1 at concentrations up to 100 pg/ml did not block the indirect twitch of the rat diaphragm . In the chick biventer cervicis muscle, the amplitude of indirect twitch was decreased to about 15%,then gradually recovered to 80% of the pre-toxin level, by 30pg/ml toxin, and it was completely blocked by 1001rg/ml toxin at 10 min. The toxin (30 pg/ml) blocked the contracture induced by 30 pg/ml ACh, but not that induced by high KCI (30 mM) . It is wncluded that respiratory failure resulting from neuromuscular block is the primary cause of death due to waglerin-1 in mice and that rats are rather resistant to waglerin-1 . Thus, waglerin-I is a novel type neurotoxin in snake venoms . The views of the author do not purport to reflect the positions of the Department of the Army or the Department of Defense. The experiments conducted therein were performed aceording to the principles set forth in the current edition of the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council. Bothrojaracin, a potent and speck thrombin inhibitor from Bothrops jararaca venom . R. Ztxont.t,'~ M . JANDAOT-PERRUS, 3 M .-C. Gtnt.uN' and C. Horn' (' Unité des Venins, Institut Pasteur, Paris, France ; ZDepartamento do Bioquimica, Universidade Federal do Rio de Janeiro, Brazil; and'Hémostase et Thrombose, Faculté Xavier Bichat, Université Paris VII, Paris, France). A Ttntouumtx specific inhibitor, named bothrojaracin, has been identified in the venom of Bothrops jararaca and purified to homogeneity . It is an acidic protein of mol . wt 27,000 and is composed of two disulfide-linked subunits of 13,500 and 15,000 . It has some structural similarities with botrocetin, a component characterised in the same venom and which binds to von Willebrand factor and induces platelet agglutination . Bothrojaracin forms an equimolecular non~ovalent complex with human a-thrombin ; however, it does not affect the catalytic site of thrombin, since amidolytic activity, measured with a chromogenic substrate S-2238, remained unchanged in the