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Genomes of Viperidae snakes contain Artiodactyla specific ART-2 retroposon
Abstracts
310
NQl and WCljl) grown under identical culture conditions differ markedly with ciguatoxin precursors detected onlv from the WCl/l strain. In contrast. all three strains produce maitotoxins. However, the FP and NQI strains produce large maitotoxins (>3000 mol.‘wt), whereas the-WCljl strain produces a small maitotoxin (mol. wt of 1060 for the disodium salt). Strain-dependent toxin production also occurs with the benthic dinoflagellate Coolia monofis. Coolia monotis was previously thought to be a non-toxic species. However, a strain of C. monotis isolated from Platypus Bay in Australia was found to produce a new toxin that was named cooliatoxin. Cooliatoxin has a mol. wt of 1062 for the sodium salt and an intraperitoneal LD~~ of 1 mg. kg-’ in mice. It induces hypothermia and respiratory failure in mice after a pronounced delay period during which there are no obvious signs of intoxication. These bioassay signs have not been reported from toxic fish extracts, and therefore there is no evidence to suggest that cooliatoxin accumulates in fish to cause human poisoning.
ABSTRACTS
OF POSTER
SESSIONS
Genomes of Viperidae snakes contain Artiodactyla specific ART-2 retroposon. D. KordiS and F. GubenSek (Department of Biochemistry and Molecular Biology, Joief Stefan Institute, Jamova 39, 61111 Ljubljana, Slovenia). Comparison of highly conserved intron sequences of Viperidae venom phospholipase A, (PLA,) genes revealed that ammodytoxin C and ammodytin L genes contain in the 4th intron nearly the same 630 bp retroposon sequence. The sequence is 75% identical to Arfiodacryla consensus ART-2 retroposon. It is widely dispersed in the genome of Vipera ammodytes, as confirmed by Southern blot analysis. The nucleotide sequences of ART-2 retroposon in both PLA, genes differ by only 2.4%. The detection of an ART-2 retroposon in V. ammodytes genome in addition to genomes of family Bovidae disagree with the present understanding of SINE distribution (D. KordiS and F. Gubengek, Nature Genetics 1995, in press). To examine a possible presence of the ART-2 retroposon in other vertebrate classes, we performed a Southern blot analysis using genomic DNA from the members of four vertebrate classes Mammalia, Aves, Reptiha, Amphibia and Arthropoda (tick Lodes ricinus). This experiment has shown that in tested Mammalia ART-2 retroposon is not present outside the family Bovidae, neither is it present in chicken, lizard, frog or tick genomes. It is, however, present in the genomes of all three tested members of Viperinae (V. ammodytes, V. palestinae, Echis coloratus) and Crotalinae (Bothrops alternatus) subfamilies. These results may indicate that the amplification of ART-2 retroposon in snakes occurred before the divergence of Viperinae and Crotalinae subfamilies. The horizontal transfer thus seems to be the only possible explanation of the origin of this retroposon in V. ammodytes and other viperid snakes. Effects of crotoxin on murine omohyoid muscle: spectrofluorimetry, Jluorescence imaging, and creatine kinase release. P. A. Melo,‘, C. F. Burns,2 C. L. Ownby’ and J. T. Blankemeyer2 (‘Departments of Physiological Sciences and ‘Zoology, Oklahoma State University, Stillwater, OK 74078, U.S.A.; and ‘Departamento de Farmacologia Basica e Clinica, ICB, UFRJ, Rio de Janeiro, RJ, Brazil). To study the myotoxicity of crotoxin (CTX), we used an electrochromic fluorescent dye, DI-4-ANEPPS (Di-4), to measure the changes in membrane potential of murine omohyoid muscle. Before CTX (10 pg/ml) was added to the medium, the muscle was incubated for 30 min in physiologic saline (PSS) containing 1 pM of Di-4. The preparation was illuminated using a dual-wavelength spectrofluorimeter (PTI), and the Di-4 fluorescence was recorded at 1 min intervals or continuously for IO-20 min. CTX caused a significant depolarization, relative to the negative controls, within 60 set of exposure. Depolarization by valinomycin (1 PM in a 100 mM KCl-enriched PSS) served as a positive control. The same preparation was examined with a fluorescence imaging system. Within 3 min of CTX exposure, fluorescence images revealed muscle contractions, bright spots along the fibres and differing sensitivity to CTX between muscle bundles. The quantitative fluorescence data agreed with the PTI results. We also measured the increase in creatine kinase (CK) release above the basal level. CTX (10 pg/ml) caused an increase in CK release from a basal level of 0.36 k 0.10 U/g/hr (N = 6) to 4.82 _+ 1.70U/g/hr (N = 6) after 10 min. Paulo
A. Melo has a CNPq
Postdoctoral
Fellowship
The effect of antibodies directed against the crotoxin on the frog nerve-muscle preparation. I. Horvat-ZnidarSi&’ V. Choumet,2 C. Bon* and D. &put’ (‘Institute of Pathophysiology, School of Medicine, P.O. Box 11, Ljubljana; and 2Unite des Venins, Institut Pasteur, 25 rue du Dr Roux, Paris Cedex 15, France). Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terriJicus, which blocks neuromuscular transmission at a presynaptic level. The crotoxin molecule is composed of a basic phospholipase A,, called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit, called
310
NQl and WCljl) grown under identical culture conditions differ markedly with ciguatoxin precursors detected onlv from the WCl/l strain. In contrast. all three strains produce maitotoxins. However, the FP and NQI strains produce large maitotoxins (>3000 mol.‘wt), whereas the-WCljl strain produces a small maitotoxin (mol. wt of 1060 for the disodium salt). Strain-dependent toxin production also occurs with the benthic dinoflagellate Coolia monofis. Coolia monotis was previously thought to be a non-toxic species. However, a strain of C. monotis isolated from Platypus Bay in Australia was found to produce a new toxin that was named cooliatoxin. Cooliatoxin has a mol. wt of 1062 for the sodium salt and an intraperitoneal LD~~ of 1 mg. kg-’ in mice. It induces hypothermia and respiratory failure in mice after a pronounced delay period during which there are no obvious signs of intoxication. These bioassay signs have not been reported from toxic fish extracts, and therefore there is no evidence to suggest that cooliatoxin accumulates in fish to cause human poisoning.
ABSTRACTS
OF POSTER
SESSIONS
Genomes of Viperidae snakes contain Artiodactyla specific ART-2 retroposon. D. KordiS and F. GubenSek (Department of Biochemistry and Molecular Biology, Joief Stefan Institute, Jamova 39, 61111 Ljubljana, Slovenia). Comparison of highly conserved intron sequences of Viperidae venom phospholipase A, (PLA,) genes revealed that ammodytoxin C and ammodytin L genes contain in the 4th intron nearly the same 630 bp retroposon sequence. The sequence is 75% identical to Arfiodacryla consensus ART-2 retroposon. It is widely dispersed in the genome of Vipera ammodytes, as confirmed by Southern blot analysis. The nucleotide sequences of ART-2 retroposon in both PLA, genes differ by only 2.4%. The detection of an ART-2 retroposon in V. ammodytes genome in addition to genomes of family Bovidae disagree with the present understanding of SINE distribution (D. KordiS and F. Gubengek, Nature Genetics 1995, in press). To examine a possible presence of the ART-2 retroposon in other vertebrate classes, we performed a Southern blot analysis using genomic DNA from the members of four vertebrate classes Mammalia, Aves, Reptiha, Amphibia and Arthropoda (tick Lodes ricinus). This experiment has shown that in tested Mammalia ART-2 retroposon is not present outside the family Bovidae, neither is it present in chicken, lizard, frog or tick genomes. It is, however, present in the genomes of all three tested members of Viperinae (V. ammodytes, V. palestinae, Echis coloratus) and Crotalinae (Bothrops alternatus) subfamilies. These results may indicate that the amplification of ART-2 retroposon in snakes occurred before the divergence of Viperinae and Crotalinae subfamilies. The horizontal transfer thus seems to be the only possible explanation of the origin of this retroposon in V. ammodytes and other viperid snakes. Effects of crotoxin on murine omohyoid muscle: spectrofluorimetry, Jluorescence imaging, and creatine kinase release. P. A. Melo,‘, C. F. Burns,2 C. L. Ownby’ and J. T. Blankemeyer2 (‘Departments of Physiological Sciences and ‘Zoology, Oklahoma State University, Stillwater, OK 74078, U.S.A.; and ‘Departamento de Farmacologia Basica e Clinica, ICB, UFRJ, Rio de Janeiro, RJ, Brazil). To study the myotoxicity of crotoxin (CTX), we used an electrochromic fluorescent dye, DI-4-ANEPPS (Di-4), to measure the changes in membrane potential of murine omohyoid muscle. Before CTX (10 pg/ml) was added to the medium, the muscle was incubated for 30 min in physiologic saline (PSS) containing 1 pM of Di-4. The preparation was illuminated using a dual-wavelength spectrofluorimeter (PTI), and the Di-4 fluorescence was recorded at 1 min intervals or continuously for IO-20 min. CTX caused a significant depolarization, relative to the negative controls, within 60 set of exposure. Depolarization by valinomycin (1 PM in a 100 mM KCl-enriched PSS) served as a positive control. The same preparation was examined with a fluorescence imaging system. Within 3 min of CTX exposure, fluorescence images revealed muscle contractions, bright spots along the fibres and differing sensitivity to CTX between muscle bundles. The quantitative fluorescence data agreed with the PTI results. We also measured the increase in creatine kinase (CK) release above the basal level. CTX (10 pg/ml) caused an increase in CK release from a basal level of 0.36 k 0.10 U/g/hr (N = 6) to 4.82 _+ 1.70U/g/hr (N = 6) after 10 min. Paulo
A. Melo has a CNPq
Postdoctoral
Fellowship
The effect of antibodies directed against the crotoxin on the frog nerve-muscle preparation. I. Horvat-ZnidarSi&’ V. Choumet,2 C. Bon* and D. &put’ (‘Institute of Pathophysiology, School of Medicine, P.O. Box 11, Ljubljana; and 2Unite des Venins, Institut Pasteur, 25 rue du Dr Roux, Paris Cedex 15, France). Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terriJicus, which blocks neuromuscular transmission at a presynaptic level. The crotoxin molecule is composed of a basic phospholipase A,, called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit, called