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Structure and function of toxins from Central Asia arachnid
Ninth European Symposium
28 5
cells. Thus, differentiation, characterized by the induction of a neuron like morphology, is accompanied by the expression of receptor molecules for the toxin . Our interest has been particularly focused on the modulation of the receptor and immunochemical detection of proteins that are secreted in response to toxin, modulated by growth factors in this and other cell systems . The conditions and duration of this response have been studied; each of these processes will be discussed. Structure and function of toxins from Central Asia arachnid. E. V. Gwsrnrr (Shemyakin institute of Bioorganic
Chemistry, U.S .S .R. Academ y of Sciences, Moscow, U.S .S.R .)
Try srwY of different toxic components from poisonous arachnid collected in Central Asia of the U.S.S .R. was undertaken . Nineteen polypeptide toxins were isolated from the venom of the Bathos eapeas scorpion, fourteen of which being toxic to mammals and five being toxic to insects. Four neurotozins active to mammals were isolated from the venom of black scorpion Orthochirus scrobicwlosus . Some structural peculiarities of scorpion toxins from Central Asia were established and amino acid sequences of nine polypeptide neurotozins were estimated. The protein toxin (M, ~ 10 kD) was isolated from the venom of tarantula Lycosa singoriensis . This toxin can increase the influx of calcium ions into the myoplasm of muscle cells. The family of high molecular weight neurotoxins (M, ~ 120-140 kD) was isolated from the venom of black widow spider Latrodectus mactans tredt."cimguttatus . All these neurotoxins were shown to be very specific for vertebrate (a-latrotoxin), for insects (latroinsectotoxins) or for crustacean (a-latrocrustatoxin) . cDNA encoding the putative a-latrotozin precursor was sequenced and the peculiarity of the toxin molecular organization was studied. A set of low molecular weight polyamine toxins blocking glutamate receptors were isolated from the venom of spiders Argiope lobata and Tegerraria dortustica. Structures of several polyamine toxins belonging to four structural types of glutamate receptor antagonists from spider venom were determined . Biochemistry and pharmacology of the big clostridia! cytotoxins. E. H~rwrtivtv, P. Berre and F. Mxut.ea
(Rudolf-Buchheim-Institut fur Pharmakologie, Justus-Liebig-Universitât, 6300-Gießen, F .R.G .)
Cwsrxrnt~ produce two types of cytotoxins acting on the cytoskeleton. The C2 toxin of C. botulirrean, the iota toxin of C. perjringens and the toxin of C. spirojorme all ADP ribosylate G-actin specifically which ultimately leads to disorganization of the cytoskeletal microfilaments . We have studied the second group consisting of C. novyi a-toxin (a-nov), C. dijßcile toxin (ß-dif) and C. sordellü toxin (LT-sor) . They are composed of single peptide chains of about 200 kDa. ß-dif and LT-sor but not a-nov cross-reacted with rabbit polyclonal antibodies . Toxicity upon i.v . injection in mice was similar (r.n~, 100 hr, 50-200 ng/kg) and characterized by a slowly developing fluid loss into the interstitial space. When injected intrawtaneously in rats the toxins caused a delayed local edema which could be monitored by volumetry of the injected rat paw for many days . The order of potency was LT-sor > a-nov > ß-dif. The three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ß-dif and a-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. Inhibition of uridine incorporation into endothelial or chicken embryonic cells reflected the cytopathic effect of the three toxins . The sequence of senitivity was ß-dif » a-nov ~ LT-sor. However, the process was a socondary event because T 84 cells responded by an about 10-fold increase of incorporation when exposed to ß-dif or a-nov but not to LT-sor. Neither toxin ADP-ribosylated actin. Notwithstanding the qualitative and quantitative differences mentioned, the numerous similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the miaofilament system of the cells in a still unknown way. Changes in Von Willebrand factor in patients bitten by Bothrops snakes . Aura S. K~Qrm, R. A. Hrrrrox,
K~rt~rxe H. Merrrmws, Ine S. S~xo-Mex'rtNS,' J. L. C. Cexnosoz and R. D. G. Trm.~r~sronr3 (Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, U.K ., 'Haematology Dept and Nits! Brazil Hospital, Institute Batsman, Sâo Paulo, Brazil and'Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, U.K.) . Htn~t~xs bitten by snakes of the genus Bothrops frequently develop severe coagulopathy . In this study, 15/27 patients attending the Vital Brazil Hospital, Insitute Batsman, Säo Paulo, following B. jararaca bite, had incoagulable blood. The majority of those tested, with or without defibrination, also had elevated levels of von Willebrand factor (vWF), measured by ELISA (meanf 2 S.E .M .: 217 f 12 .0 U/dl, range: 193-232 U/dl). Levels in local control subjects (n = 10) were 40-168 U/dl (meanf 2 S.E .M . 92 .7 f 23 .4 U/dl). Incubation of normal citrated plasma with 200 pg/ml of B. jararaca venom for 15 min at 37°C produced an apparent increase in vWF measured by Laurell electroimmunoassay (EIA) from 88.3 f5.4 U/dl to 165.4 f 12.2 U/dl, but not by ELISA (pre (00.2 f4 .0 U/dl, post 110 f3.4 U/dl) . This suggests that the venom cleaves vWF producing lower M, forms with increaved electrophoretic mobility though retaining their antigenicity. Multimeric analysis supported this interpretation, showing only low M, forms of vWF in these samples.
28 5
cells. Thus, differentiation, characterized by the induction of a neuron like morphology, is accompanied by the expression of receptor molecules for the toxin . Our interest has been particularly focused on the modulation of the receptor and immunochemical detection of proteins that are secreted in response to toxin, modulated by growth factors in this and other cell systems . The conditions and duration of this response have been studied; each of these processes will be discussed. Structure and function of toxins from Central Asia arachnid. E. V. Gwsrnrr (Shemyakin institute of Bioorganic
Chemistry, U.S .S .R. Academ y of Sciences, Moscow, U.S .S.R .)
Try srwY of different toxic components from poisonous arachnid collected in Central Asia of the U.S.S .R. was undertaken . Nineteen polypeptide toxins were isolated from the venom of the Bathos eapeas scorpion, fourteen of which being toxic to mammals and five being toxic to insects. Four neurotozins active to mammals were isolated from the venom of black scorpion Orthochirus scrobicwlosus . Some structural peculiarities of scorpion toxins from Central Asia were established and amino acid sequences of nine polypeptide neurotozins were estimated. The protein toxin (M, ~ 10 kD) was isolated from the venom of tarantula Lycosa singoriensis . This toxin can increase the influx of calcium ions into the myoplasm of muscle cells. The family of high molecular weight neurotoxins (M, ~ 120-140 kD) was isolated from the venom of black widow spider Latrodectus mactans tredt."cimguttatus . All these neurotoxins were shown to be very specific for vertebrate (a-latrotoxin), for insects (latroinsectotoxins) or for crustacean (a-latrocrustatoxin) . cDNA encoding the putative a-latrotozin precursor was sequenced and the peculiarity of the toxin molecular organization was studied. A set of low molecular weight polyamine toxins blocking glutamate receptors were isolated from the venom of spiders Argiope lobata and Tegerraria dortustica. Structures of several polyamine toxins belonging to four structural types of glutamate receptor antagonists from spider venom were determined . Biochemistry and pharmacology of the big clostridia! cytotoxins. E. H~rwrtivtv, P. Berre and F. Mxut.ea
(Rudolf-Buchheim-Institut fur Pharmakologie, Justus-Liebig-Universitât, 6300-Gießen, F .R.G .)
Cwsrxrnt~ produce two types of cytotoxins acting on the cytoskeleton. The C2 toxin of C. botulirrean, the iota toxin of C. perjringens and the toxin of C. spirojorme all ADP ribosylate G-actin specifically which ultimately leads to disorganization of the cytoskeletal microfilaments . We have studied the second group consisting of C. novyi a-toxin (a-nov), C. dijßcile toxin (ß-dif) and C. sordellü toxin (LT-sor) . They are composed of single peptide chains of about 200 kDa. ß-dif and LT-sor but not a-nov cross-reacted with rabbit polyclonal antibodies . Toxicity upon i.v . injection in mice was similar (r.n~, 100 hr, 50-200 ng/kg) and characterized by a slowly developing fluid loss into the interstitial space. When injected intrawtaneously in rats the toxins caused a delayed local edema which could be monitored by volumetry of the injected rat paw for many days . The order of potency was LT-sor > a-nov > ß-dif. The three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ß-dif and a-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. Inhibition of uridine incorporation into endothelial or chicken embryonic cells reflected the cytopathic effect of the three toxins . The sequence of senitivity was ß-dif » a-nov ~ LT-sor. However, the process was a socondary event because T 84 cells responded by an about 10-fold increase of incorporation when exposed to ß-dif or a-nov but not to LT-sor. Neither toxin ADP-ribosylated actin. Notwithstanding the qualitative and quantitative differences mentioned, the numerous similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the miaofilament system of the cells in a still unknown way. Changes in Von Willebrand factor in patients bitten by Bothrops snakes . Aura S. K~Qrm, R. A. Hrrrrox,
K~rt~rxe H. Merrrmws, Ine S. S~xo-Mex'rtNS,' J. L. C. Cexnosoz and R. D. G. Trm.~r~sronr3 (Haemophilia Centre and Haemostasis Unit, Royal Free Hospital, London, U.K ., 'Haematology Dept and Nits! Brazil Hospital, Institute Batsman, Sâo Paulo, Brazil and'Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, U.K.) . Htn~t~xs bitten by snakes of the genus Bothrops frequently develop severe coagulopathy . In this study, 15/27 patients attending the Vital Brazil Hospital, Insitute Batsman, Säo Paulo, following B. jararaca bite, had incoagulable blood. The majority of those tested, with or without defibrination, also had elevated levels of von Willebrand factor (vWF), measured by ELISA (meanf 2 S.E .M .: 217 f 12 .0 U/dl, range: 193-232 U/dl). Levels in local control subjects (n = 10) were 40-168 U/dl (meanf 2 S.E .M . 92 .7 f 23 .4 U/dl). Incubation of normal citrated plasma with 200 pg/ml of B. jararaca venom for 15 min at 37°C produced an apparent increase in vWF measured by Laurell electroimmunoassay (EIA) from 88.3 f5.4 U/dl to 165.4 f 12.2 U/dl, but not by ELISA (pre (00.2 f4 .0 U/dl, post 110 f3.4 U/dl) . This suggests that the venom cleaves vWF producing lower M, forms with increaved electrophoretic mobility though retaining their antigenicity. Multimeric analysis supported this interpretation, showing only low M, forms of vWF in these samples.