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The African vipers Echis ocellatus have a selective predation, possibly influencing their encounter with man
Eleventh European Mating
40 5
has the potential to provide large amounts of recombinant XTx for further biochemical and toxicological analysis . Fxor~tert et al. (1988) PNAS ~, 8998-9002. Detection oJriciit by colorimetric and chemilumüeescence ELISA . M. A. Poly' V. R. RrveRw,' J. F. Iiswsrsort' and
G. A. M~rwu.2 ('Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, U.S .A .; and'Brooke Army Medical Center, San Antonio, TX, U.S .A .) . A Enaro.Y sensitive and specific calorimetric ELISA was developed to detect ricin in biological fluids. The assay utilizes a high amity (Kd = 10 - '°-10- " M) goat polyclonal antibody to adsorb ricin from solution . Biotinylated antibody is then used to form a sandwich, and avidin-linked alkaline phoaphatase allows color development and measurement of optical density at 405 nm . The standard assay utilizes a standard curve over the range of 1-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in our assay buffer as well as in 1 :10 human urine and 1 : 50 human serum. Measuring ricin in spiked samples (n = ~ at three different cancentration points within the standard curve demonstrated accuracy typically within 5% of the expected value (range = 0-12%) . Accuracy was not affectod by matrix . Coefficient of variation ringed from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml . Two variations on the standard calorimetric assay were also investigated . First, lengthening incubation times and allowing additional time for color development allowod accurate quantitation of ricin in serum dilutions as low as 1 :2 . Second, increasing the concentrations of biotinylated second antibody and avidin-linked alkaline phosphatase from 1 :250 to 1:70 increased the sensitivity of the assay by an order of magnitude, to at kast 100 pg/ml (10 pg/well) . Additional sensitivity could likely be achieved through additional manipulation . We modified this assay into a chemiluminescence format with Lumi-Phos 530 (Lumigen, Inc.) as substrate. This chemiluminescence format allowed accurate quantitation in the 0 .1-1 ng/ml range, but was subject to slightly greater day-today variability than the calorimetric assay. The assays described here are the most sensitive for ricin reported to date and are at kast 10-50-fold more sensitive than other ricin ELISAs . Further, the high sensitivity and accuracy of these assays in serum should support in vfvo distribution studies. 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 herein 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. Cloning and sequencing of cDNNs encoding tquinatoxins from the European sea mremoru Actinic equine. J. Puxo>:a~re,' B. $rxurv:u,' P. Mr,C~Z and F. Gtr' ('Department of Biochemistry and Molecular Biology, Jo~ef Stefan Institute, Jamova 39, P.O . Box 100, 61111 Ljubljana, Slovenia ; and =Department of Biochemistry, Biotechnical Faculty, University of Ljubljana, 61000 Ljubljana, Slovenia). Srr+e~tot~s possess a number of biologically active Compounds including some potent toxic polypeptides and proteins. One of the more characterized classes of these toxins are membrane-active cytolysins which are basic proteins devoid of cysteine residues with molecular mass in the range 15,000 to 20,000 . Cytolysins with similar cardiatic stimulatory and haemolytic activities were found in different species of sea anemones, such as Actinic equine, A . tenebrosa, A . cmi and Stichodacryle helianthus. On the basis of nearly perfect protein sequence identity between equinatoxin II from the European sea anemone A . equine and tenebrosin-C from the Australian red warath sea anemone A . torebrasc and almost identical biological activities, it has been suggested that these two anemones represent closely related species or subspecies . In order to get more information about the taxonomic relationship between these two species of sea anemones and to get more insight into the not yet Completely understood mode of action of these interesting membrane-active cytotoxins, we started with molecular cloning of complementary DNA (cDNA) of equinatoxins from A . equine. Total RNA was isolated from a single sea anemone specimen and poly(A)+ RNA separated on an oligo(dT}cellulose column . The first strand of cDNA was synthesized by reverse tranacriptase and amplified by a polymerise chain reaction (PCR) using two mixed oligonucleotide primers with degeneracy of 128 and 1024, respectively, deducod from a part of the primary structure of equinatoxin II . A cDNA library in bacteriophage lambda gtl l was prepared from the poly(A)+ RNA and screened for positive clones using the positive PCR product of about 300 bp, encoding a part of the equinatoxin cDNA sequence, as a probe. Positive cDNA clones were subcloned into a plasmid vector and Complete proton sequences of equinatoxins deducod from their cDNA sequences. Nucleotide sequencing of two different cDNA Clones revealed that they Code for equinatoxin II and for a new equinatoxin showing most similarities with the N-terminal sequence of tenebrosin-B from A . terttbrosa, respectively. Their cDNA sequences indicate that these proteins are probably synthesized as larger precursor molecules, in which their mature protein sequence is preceded by a relatively hydrophobic signal peptide and a more hydrophilic propeptide . The Afrkan vipers Echis ocellatus hase a selective predetion, possibly inJheencing their encounter with man. P .
Goyffon), Muséum national d'Histoire naturelle, 57, rue Cuvier, F-75005 Paris, France). IN rte Sudan and Sahelian Savanna, vipers from the genus Echis Contribute largely to human enveaomation (Hnara, 1992; ROUteN, 1968). Predation plays a major role in the dynamic and the organization of the tropical REVAULT (L .E.R .A .L : (M .
406
Eleventh European Mating
reptile population (BARBAULT, 1991). Therefore it is interesting to know the diet of Echis . Forty-seven Echls ocdlatus Stemmler 1970 were caught in October 1992 (39 individuals) and in April 1993 (eight individuals), in four areas divided around Ouagadougou (Burkina Faso, West Africa), preservedin formalin 10%,then dissected and conserved in alcoho160% . Eight individuals did not have any digestive content. Amongst the remaining 39 vipers, 34 centipedes (genus Scolopendra, nine identified es Scolopendra morsirans Linnaeus, 1758) were discovered, and 16 small rodents (of which three were classified as Nannomys ap.) . But l9 snakes contained scolopendra, and 12 had captured rodents. In three cases the snake had swallowed one rodent and one centipede. Equally found were the remains of three snakes (one probably Psammophis sp.), one battacian (Bujo sp.), two tamarin lea$ets (Tamarindus indica), I Solifugae, 1 Coleoptera, throe ants and the remains of non-identifiable arthropods . There was no correlation between the size (or the sex) on the Echis (average length 35 cm) and the type of prey found . The predation of rodents by vipers is well known (Roatxx, 1975; Vt r r~~, 197 . Thie explains a reason why there can be found Ophidia around the houses and in the fields . It is also well known that vipers feed from other reptiles, batraciens and insects . But, to our knowledge, capture of centipedes as common prey has not been remarked up to now; even if Vu.t.rnxs (1975) sights the predation of the Myriapoda by the genus Typhlops and Leptotyphlops, both burrowing snakes. In South Africa, Colubridae from the genus .lsparallactus are described as centipede eaters. The Scoiopendra morsitans, lucifugal and needing moisture, frequently found in habitations, might explain the frequency of man-snake encounters in the night, dusk and dawn, in or around houses, principally at the beginning and the end of the rainy season. The venin of the genus Echis is essentially hemorrhagiparous, very effective for the predation of small rodents. It can be asked under what type of conditions did the predation of centipedes develop in such a characteristic way. Recently, in the genus Echis, was discovered a supralabial gland with an external aperture (Ixercx and Teca.mte, 1992), being unique amongst snakes . This could perhaps lead to interesting answers. In order to develop this study, we must experience many complete pluviometrical cycles, to look at other regions of the Sudan and Sahel Savamra (Nigeria), examining older species of the genus Echis, enabling us to enrich the comprehension of the biocenosis and the interaction between man and this environment . Structwe-activity analysis ojleiurotoxin I using synthetic analogues . J:M. S~rmx,' V. FxF.acoxr,' K. M~rtoutc,'
M. Cn>=tr ; H. DNeeoN,' H. Roct~r,' J. Vuv RterscxoT~' and M:F. M~e~ruv-Fr+ucwxe' ('Laboratoiro de Biochimie, CNRS URA 1455, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cédex 20, France; 'Laboratoire de Neurobiologie, CNRS, 31, Chemin Joseph Aiguier, 13402 Marseille Cédex 9, France ; and 'Laboratoire de Cristallographie et Cristallisation des Macromolécules Biologiques, CNRS URA 1296, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cédex 20, France). R~Tt .Y, we reported a structure activity relationship study on POS, a novel leiurotoxin I-like scorpion toxin which is selective for the apamin-sensitive Ca'+-activated K+ channel (Seaerrea et al., 1993). Argb, Arg and C-terminal His3, appeared to be key residues for POS biological activity. Owing to high sequence identity between POS and leiurotoxin I (87%), several analogs of leiurotoxin I (Lei-NH2) with point mutations at these positions were designed and chemically synthesized using optimized solid phase technique. The synthesized peptides were [L6] Lei-NH2, [R,] Lei-NH2, Lei-0H, and (R,] Lei-OH, as well as fragment IR,,Abus] N4-Sll-NH2. A chimeric analog QM~,K~ Rn] Lei-NH2), which possesses part of the iberiotoxin Gterminus, was also constructed . Circular dichroism analyses of these analogs, in agreement with their structural models obtained by molecular dynamics, ahowod that the point mutations did not significantly affect the overall secondary structures, as compared to natural Lei-NH2. All the peptides and natural toxins were compared in vitro for their capacity to inhibit binding of ['xsll-apamin to rat brain synaptosomea, and in oitw for their specific neurotoxicity in mice . The Arg° residue was essential for high biological activity of leiurotoxin I. Further, substitution of Met, in the natural toxin by Arg or C-terminal amidation of His3 greatly increased.afiinity for the apamin receptor but did not affect significantly toxin neurotoxicity . Remarkably, the chimeric analog [M~,K~ Rn] Lei-NH2 was found to retain leiurotoxin I-like activity, thus indicating that the negatively charged residues Asps, and Glur, (and Ilea) are not directly involved in the high toxin bioactivity. However, the chimeric molecule had no iberiotoxin-like effect on rat muscular maxi-K+ channels incorporated in lipid bilayers . Seeern:a, J. et al. (1993) Biochemistry 32, 2763-2770. Study on POS, a new leiurotoxin I scorpion toxin . J:M. Seaem~n,' H. ZEreaoue,r H. DetteoN,' K. Mesxoutc,' A.
Bevsr.reu~,z H. Rooter,' M:F. Mexruv-Eeucurne' and J. Very Rmrxx~rrorer~' ('CNRS URA 1455, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cédex 20, France; Institut Pasteur du Maroc, 1, Place Charles Nicolle, B.P . 120, Casablanca, Morocco; and'CNRS URA 1296, Faculté de Médecine Nord, Bd Pierre Dramerd, 13326 Marseille Cédex IS) . Tt~ v~oss of the scorpion .lndroctonus mauretanicus mauretanicus contains a toxin, POS, which is structurally and functionally similar to scorpion leiurotoxin I (87% sequence identity), a Mocker of the apamin-sensitive Cat+-activated K+ channels. It is a 3I-residue polypeptide cross-linked by three disulfide bridges. A C-terminal carboxyl-amidated analog of POS (sP05-NHr) was chemically synthesized by the solid phase technique and fully characterized . Toxicity assays R vivo established that sP05-NHz, like native POS, is a potent andlethal neurotoxic agent in mice (~so of 20 ng per mouse) . Pharmacological assays fn vitro showed, however, that unlike POS which
40 5
has the potential to provide large amounts of recombinant XTx for further biochemical and toxicological analysis . Fxor~tert et al. (1988) PNAS ~, 8998-9002. Detection oJriciit by colorimetric and chemilumüeescence ELISA . M. A. Poly' V. R. RrveRw,' J. F. Iiswsrsort' and
G. A. M~rwu.2 ('Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, U.S .A .; and'Brooke Army Medical Center, San Antonio, TX, U.S .A .) . A Enaro.Y sensitive and specific calorimetric ELISA was developed to detect ricin in biological fluids. The assay utilizes a high amity (Kd = 10 - '°-10- " M) goat polyclonal antibody to adsorb ricin from solution . Biotinylated antibody is then used to form a sandwich, and avidin-linked alkaline phoaphatase allows color development and measurement of optical density at 405 nm . The standard assay utilizes a standard curve over the range of 1-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in our assay buffer as well as in 1 :10 human urine and 1 : 50 human serum. Measuring ricin in spiked samples (n = ~ at three different cancentration points within the standard curve demonstrated accuracy typically within 5% of the expected value (range = 0-12%) . Accuracy was not affectod by matrix . Coefficient of variation ringed from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml . Two variations on the standard calorimetric assay were also investigated . First, lengthening incubation times and allowing additional time for color development allowod accurate quantitation of ricin in serum dilutions as low as 1 :2 . Second, increasing the concentrations of biotinylated second antibody and avidin-linked alkaline phosphatase from 1 :250 to 1:70 increased the sensitivity of the assay by an order of magnitude, to at kast 100 pg/ml (10 pg/well) . Additional sensitivity could likely be achieved through additional manipulation . We modified this assay into a chemiluminescence format with Lumi-Phos 530 (Lumigen, Inc.) as substrate. This chemiluminescence format allowed accurate quantitation in the 0 .1-1 ng/ml range, but was subject to slightly greater day-today variability than the calorimetric assay. The assays described here are the most sensitive for ricin reported to date and are at kast 10-50-fold more sensitive than other ricin ELISAs . Further, the high sensitivity and accuracy of these assays in serum should support in vfvo distribution studies. 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 herein 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. Cloning and sequencing of cDNNs encoding tquinatoxins from the European sea mremoru Actinic equine. J. Puxo>:a~re,' B. $rxurv:u,' P. Mr,C~Z and F. Gtr' ('Department of Biochemistry and Molecular Biology, Jo~ef Stefan Institute, Jamova 39, P.O . Box 100, 61111 Ljubljana, Slovenia ; and =Department of Biochemistry, Biotechnical Faculty, University of Ljubljana, 61000 Ljubljana, Slovenia). Srr+e~tot~s possess a number of biologically active Compounds including some potent toxic polypeptides and proteins. One of the more characterized classes of these toxins are membrane-active cytolysins which are basic proteins devoid of cysteine residues with molecular mass in the range 15,000 to 20,000 . Cytolysins with similar cardiatic stimulatory and haemolytic activities were found in different species of sea anemones, such as Actinic equine, A . tenebrosa, A . cmi and Stichodacryle helianthus. On the basis of nearly perfect protein sequence identity between equinatoxin II from the European sea anemone A . equine and tenebrosin-C from the Australian red warath sea anemone A . torebrasc and almost identical biological activities, it has been suggested that these two anemones represent closely related species or subspecies . In order to get more information about the taxonomic relationship between these two species of sea anemones and to get more insight into the not yet Completely understood mode of action of these interesting membrane-active cytotoxins, we started with molecular cloning of complementary DNA (cDNA) of equinatoxins from A . equine. Total RNA was isolated from a single sea anemone specimen and poly(A)+ RNA separated on an oligo(dT}cellulose column . The first strand of cDNA was synthesized by reverse tranacriptase and amplified by a polymerise chain reaction (PCR) using two mixed oligonucleotide primers with degeneracy of 128 and 1024, respectively, deducod from a part of the primary structure of equinatoxin II . A cDNA library in bacteriophage lambda gtl l was prepared from the poly(A)+ RNA and screened for positive clones using the positive PCR product of about 300 bp, encoding a part of the equinatoxin cDNA sequence, as a probe. Positive cDNA clones were subcloned into a plasmid vector and Complete proton sequences of equinatoxins deducod from their cDNA sequences. Nucleotide sequencing of two different cDNA Clones revealed that they Code for equinatoxin II and for a new equinatoxin showing most similarities with the N-terminal sequence of tenebrosin-B from A . terttbrosa, respectively. Their cDNA sequences indicate that these proteins are probably synthesized as larger precursor molecules, in which their mature protein sequence is preceded by a relatively hydrophobic signal peptide and a more hydrophilic propeptide . The Afrkan vipers Echis ocellatus hase a selective predetion, possibly inJheencing their encounter with man. P .
Goyffon), Muséum national d'Histoire naturelle, 57, rue Cuvier, F-75005 Paris, France). IN rte Sudan and Sahelian Savanna, vipers from the genus Echis Contribute largely to human enveaomation (Hnara, 1992; ROUteN, 1968). Predation plays a major role in the dynamic and the organization of the tropical REVAULT (L .E.R .A .L : (M .
406
Eleventh European Mating
reptile population (BARBAULT, 1991). Therefore it is interesting to know the diet of Echis . Forty-seven Echls ocdlatus Stemmler 1970 were caught in October 1992 (39 individuals) and in April 1993 (eight individuals), in four areas divided around Ouagadougou (Burkina Faso, West Africa), preservedin formalin 10%,then dissected and conserved in alcoho160% . Eight individuals did not have any digestive content. Amongst the remaining 39 vipers, 34 centipedes (genus Scolopendra, nine identified es Scolopendra morsirans Linnaeus, 1758) were discovered, and 16 small rodents (of which three were classified as Nannomys ap.) . But l9 snakes contained scolopendra, and 12 had captured rodents. In three cases the snake had swallowed one rodent and one centipede. Equally found were the remains of three snakes (one probably Psammophis sp.), one battacian (Bujo sp.), two tamarin lea$ets (Tamarindus indica), I Solifugae, 1 Coleoptera, throe ants and the remains of non-identifiable arthropods . There was no correlation between the size (or the sex) on the Echis (average length 35 cm) and the type of prey found . The predation of rodents by vipers is well known (Roatxx, 1975; Vt r r~~, 197 . Thie explains a reason why there can be found Ophidia around the houses and in the fields . It is also well known that vipers feed from other reptiles, batraciens and insects . But, to our knowledge, capture of centipedes as common prey has not been remarked up to now; even if Vu.t.rnxs (1975) sights the predation of the Myriapoda by the genus Typhlops and Leptotyphlops, both burrowing snakes. In South Africa, Colubridae from the genus .lsparallactus are described as centipede eaters. The Scoiopendra morsitans, lucifugal and needing moisture, frequently found in habitations, might explain the frequency of man-snake encounters in the night, dusk and dawn, in or around houses, principally at the beginning and the end of the rainy season. The venin of the genus Echis is essentially hemorrhagiparous, very effective for the predation of small rodents. It can be asked under what type of conditions did the predation of centipedes develop in such a characteristic way. Recently, in the genus Echis, was discovered a supralabial gland with an external aperture (Ixercx and Teca.mte, 1992), being unique amongst snakes . This could perhaps lead to interesting answers. In order to develop this study, we must experience many complete pluviometrical cycles, to look at other regions of the Sudan and Sahel Savamra (Nigeria), examining older species of the genus Echis, enabling us to enrich the comprehension of the biocenosis and the interaction between man and this environment . Structwe-activity analysis ojleiurotoxin I using synthetic analogues . J:M. S~rmx,' V. FxF.acoxr,' K. M~rtoutc,'
M. Cn>=tr ; H. DNeeoN,' H. Roct~r,' J. Vuv RterscxoT~' and M:F. M~e~ruv-Fr+ucwxe' ('Laboratoiro de Biochimie, CNRS URA 1455, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cédex 20, France; 'Laboratoire de Neurobiologie, CNRS, 31, Chemin Joseph Aiguier, 13402 Marseille Cédex 9, France ; and 'Laboratoire de Cristallographie et Cristallisation des Macromolécules Biologiques, CNRS URA 1296, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cédex 20, France). R~Tt .Y, we reported a structure activity relationship study on POS, a novel leiurotoxin I-like scorpion toxin which is selective for the apamin-sensitive Ca'+-activated K+ channel (Seaerrea et al., 1993). Argb, Arg and C-terminal His3, appeared to be key residues for POS biological activity. Owing to high sequence identity between POS and leiurotoxin I (87%), several analogs of leiurotoxin I (Lei-NH2) with point mutations at these positions were designed and chemically synthesized using optimized solid phase technique. The synthesized peptides were [L6] Lei-NH2, [R,] Lei-NH2, Lei-0H, and (R,] Lei-OH, as well as fragment IR,,Abus] N4-Sll-NH2. A chimeric analog QM~,K~ Rn] Lei-NH2), which possesses part of the iberiotoxin Gterminus, was also constructed . Circular dichroism analyses of these analogs, in agreement with their structural models obtained by molecular dynamics, ahowod that the point mutations did not significantly affect the overall secondary structures, as compared to natural Lei-NH2. All the peptides and natural toxins were compared in vitro for their capacity to inhibit binding of ['xsll-apamin to rat brain synaptosomea, and in oitw for their specific neurotoxicity in mice . The Arg° residue was essential for high biological activity of leiurotoxin I. Further, substitution of Met, in the natural toxin by Arg or C-terminal amidation of His3 greatly increased.afiinity for the apamin receptor but did not affect significantly toxin neurotoxicity . Remarkably, the chimeric analog [M~,K~ Rn] Lei-NH2 was found to retain leiurotoxin I-like activity, thus indicating that the negatively charged residues Asps, and Glur, (and Ilea) are not directly involved in the high toxin bioactivity. However, the chimeric molecule had no iberiotoxin-like effect on rat muscular maxi-K+ channels incorporated in lipid bilayers . Seeern:a, J. et al. (1993) Biochemistry 32, 2763-2770. Study on POS, a new leiurotoxin I scorpion toxin . J:M. Seaem~n,' H. ZEreaoue,r H. DetteoN,' K. Mesxoutc,' A.
Bevsr.reu~,z H. Rooter,' M:F. Mexruv-Eeucurne' and J. Very Rmrxx~rrorer~' ('CNRS URA 1455, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cédex 20, France; Institut Pasteur du Maroc, 1, Place Charles Nicolle, B.P . 120, Casablanca, Morocco; and'CNRS URA 1296, Faculté de Médecine Nord, Bd Pierre Dramerd, 13326 Marseille Cédex IS) . Tt~ v~oss of the scorpion .lndroctonus mauretanicus mauretanicus contains a toxin, POS, which is structurally and functionally similar to scorpion leiurotoxin I (87% sequence identity), a Mocker of the apamin-sensitive Cat+-activated K+ channels. It is a 3I-residue polypeptide cross-linked by three disulfide bridges. A C-terminal carboxyl-amidated analog of POS (sP05-NHr) was chemically synthesized by the solid phase technique and fully characterized . Toxicity assays R vivo established that sP05-NHz, like native POS, is a potent andlethal neurotoxic agent in mice (~so of 20 ng per mouse) . Pharmacological assays fn vitro showed, however, that unlike POS which