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Tarantula venom: physical attributes and chromatographic fractionation of Brachypelma emilia venom
136
Second American Symposium
in the clinically positive pony, hematology and serum chemistry were normal in all ponies . Brain pathology was consistent with ELEM only in the clinically positive pony . Cytoplasmic changes (vacuolation, clumping) were observed in hepatocytes of all animals. Routine analysis of the cerebrospinal fluid (CSF) indicated chronic inflammation in both the clinically positive and one non~linical pony . Basic myelin protein of CSF was markedly elevated in the clinically positive pony as opposed to non~linical ponies (14 ng/ml vs . CL ng/ml). The results show susceptibility of ponies to Fusarium monilS%rme toxins) and suggest that basic myelin protein could be used as a marker in this condition. Additional studies involving strains isolated from the feed suspected in the North Carolina outbreak are in progress .
Fffert of T-2 mycotaxin on amino acid uptake in L~ myablastS. DAVID L. BUNNER, ELENA R. MORRIS, JUDITH G. PwcE and CHARLES F. MATSON (U .S . Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701, U .S .A .) . PROTEIN synthesis inhibition is a well documented direct cellular effect of T-2 mycotoxin. Other effects, such as alteration in amino acid uptake, have not been assessed . Since in vivo studies have documented hyperaminoacidemia after T-2 exposure and muscle is the largest single amino acid pool, amino acid uptake in L-6 myoblasts was measured . Confluent L-6 cells were studied after both S and 60 min exp~ure to T-2 and the cellular uptake (both acid-soluble and -precipitable) of aminoisobutyric acid, leucine and tyrosine were measured after a 1 and 3 min pulse of amino acid . Protein synthesis was reduced maximally (94- 9340) at higher doses of toxin (40 and 400 ng/ml) but was leas affected (SS - 8440 at the lowest dose (4 ng/ml) . Amino acid uptake was equally reduced at all doses (34- 7540). There was no significant difference between the S and 60 min exposure regarding protein synthesis inhibition or reduction in amino acid uptake . Radioisotopic scanning of thin-layer chromatography plates showed that leas than 340 of the parent T-2 was metabolized by 60 min. Amino acid metabolism was negligible. In conclusion, T-2 reduced both protein synthesis and amino acid uptake in L-6 myoblaata. Whether or not these two effects have any causal relationship could not be determined from the data .
Advaru+es in ncsaanh on jellyfish tanins and their pathogenic mechanisms. J~erx W. BURNETT and Gwev J. CALTON (Division of Dermatology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S .A .) . RESEARCx on the chemical characteristics of several jellyfish (Chrysaora quinquerirrha and Physolia physalis) toxins has been greatly aided by affinity immunochromatography procedures . The fishing tentacle of the sea nettle (Chrysaom quirrquecirrha) contains two polypeptides of mol. wt 190,000 and 100,000. The mesentery tentacles have a nematocyst venom containing three polypeptides of mol. wt 80,000, 160,000 and 110,000. A major action of these proteins is to increase monovalent and divalent cation transport across biological membranes. This transport increase is aided by the production of large holes in the membrane and can be reversed by the use of verapamil. Studies on the nematocyst venom of Portuguese man-0'war (Physaliaphysalis) have been aided by the use of an automated all sorter . With this technique, isolated nematocyst syspensions can be obtained free of all tentacle debris . This animal has two sizes of nematocysts, (10 and 23 Fart diameter), the smaller of which was found to be the more biologically active . The most prominent protein present in that nematocyst solution was a 70,000 mol.wt band . A aoas-reactivity to antigens present in either jellyfish venom has been demonstrated in monoclonal antibodies prepared from mice and polydonal antibodies from envenomatod patients . It has been found that the more seriously envenomated victims have higher circulating titers of species-specific polyclonal antibody . Additionally, patients envenonated by either species show considerable serological cross-reactivity with other jellyfish venous . A survey of envenomated patients has revealed several syndromes produced by wntact with these jellyfish. Persistent granulomatous lesions resulting from envenomation by Pelagic noctilurn have been accompanied by abnoruial natural killer (NK) cell activities aginst Chrysaora antigen for periods up to three years after resolution of the lesion . Patients with recurrent cutaneoua lesions following a single envenomation by Physalia and other jellyfish have been described. These individuals exhibit the expected serological response to the offending animal . In addition, one patient had an immunosuppression of her T cello evidenced by lymphokine blockage during one of her recurrent eruptions. Investigations on the effect of various blockers of inflammatory mediators on the increased cutaneous vasopermeability produced by various coelenterate venoms was undertaken. The results demonstrated that no one inhibitory drug was effective against the action exhibited by all venoms . This data demonstrates the fact that future therapy of jellyfish envenomation will be species-spaific. Tarantula venom: physirnl attributes and chromatogrtirphic jractlonation of Brachypelma emilia venom. C. D. CxwsrtwN, P. D. SwwRTZ, S. A. HUDIBURG, J. R. ScxwTZ and G. V. ODELL (Department of Biochemistry, Oklahoma State University, Oklahoma Agricultural Experiment Station, Stillwater, OK 74078, U .S .A .) .
Second American Symposium
13 7
TARANTULAS have become a popular exotic pet and the flashy orange and black body and grntle manner of Brachypelma emilia make it an attractive choice . The major hazard of this spider is its unicating hairs and although the venom is not deadly to man, this increased attention makes venom characterization important. Previous work on B. emilia venom includes comparison studies performed in this laboratory by Gabbiness et. ol. The venom has a pH of about 5.65, a solids content of 125 1+g/pl and an absorbance maximum of 26l nm . Protein content will be reported . Fractionation techniques will include gel filtration, polyacrylamide gel electrophoresis, thin-layer gel filtration and high performance liquid chromatography . Purification of the toxin is a major goal of this research and characteristics of this toxin will be reported . Hyaluronidase, protease and phospholipase activities of the venom will also be presented.
lsolotion and partial characterization of two lethal toxins from prairie rattlesnake (Crotalus viridis viridis) venom. TERRY R. COLBERG end CHARLOTTE L. OWNBY (Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, U .S .A). LYOPHILIZED prairie rattlesnake (Crotalus viridis viridis) venom puchased from biotoxins was fractionated by column chromatography . Two grams of the vrnom were dissolved in 0.01 M Tris buffer, pH 8.6, and after desalting by ultrafiltration was applied to a DEAF cellulose ion exchange column . The same buffer was used initially for elution until the absorbance at 280 nm returned to baseline . Then a NaCI gradirnt (0-0 .3 M) in the Tris buffer was used to elute the remaining protein. This procedure resulted in S fractions, all of which were tested for hemorrhagic, mytoxic and lethal activities by intramuscular injection into mice . Fraction Al from this rnlumn caused myonecrosis and hemorrhage at both 30 min and 24 hr after injection . It was pooled, concentrated, equilibrated with 0.01 M MOPS buffer, pH 7.2, and applied to an ultrogel AcA34 gel filtration column. Elution with this buffer yielded 8 fractions . Fraction BS from this column was non-hemorrhaic, myotoxic and lethal . This fraction was pooled, concrntrated and applied to a Whatman CM32 anion exchange column which was eluted with a linear salt gradient (0-0 .8 M NaCI) in 0.01 M MOPS buffer, pH 7.2 . This step resulted in 7 fractions of which Fractions C3 and CS caused death within 10 min after injection of 2.9 and 1.19 pg/g, respectively . Fraction C6 from this column was myotoxic but not lethal . Preliminary rraults indicate that Fraction C3 has an LDm (i.m .) of 0.03 Ng/g and Fraction CS of 0.09 F+8/g. These two toxins are being purified and assayed for protease, esterase and phospholipase activities .
Analysis ojjatty acids in the lipid rnmponents ojsnake venom. MARY E. CooKe, GEORGE V. ODELL, SuE A. HuD~auaG, JosE M. GUTIERREZ (Departmrnt of Biochemistry, Oklahoma State University, Oklahoma Agricultural Experiment Station, Stillwater, OK 74078, U.S .A ., and Inatituto Clodimiro Picado, Department of Biochemistry, San JosE, Costa Rita). THE LIPID fraction of Crotalus atrox lyophilized venom has been extracted. The methyl ester derivatives were analyzed by gas liquid chromatography on a Perkin Elmer 990 gas chromatograph . Several fatty acids were observed . Tentative identification by G.C. retention times indicates the presrnce of aprylic, esprit, lauric and palmitic acid . A significant amount of a higher molecular weight fatty acid has also been observed . Earlier studies of the Ngja ngja venom reported a large percentage of arachadonic acid . This suggests arachadonic as a possibility for the higher molecular weight acid . Further fractionation of the lipid portion of vrnom into neutral and phospholipid components and analysis on thin layer chromatographic plates is being performed. Also, a comparison of the lipid in adipose tissue, glands and venom of the snake will be conducted .
Botulinum neurotoxin : structurr-junction and similarity to tetanus. B. R. DASGupTA, S. BANDYOPADHYAY, A. W. CLARK, A. HERIAN, V. SATHYAMOORTHY and M. A. WOODY (FOOd Research InstitotC, University Of Wisconsin, Madison, WI 33706, U.S .A .) . SIMPSON proposed (Pharmac. Rev. 33, 133) that botulinum neurotoxin (NT) binds to cholinergic nerve terminals, then a part or the entire NT molecule is internalized and finally intracellular evrnt(s) block acetylcholine release. We conceive at least 2 active sites, 1 for binding and 1 for metabolic block; damage to either site would detoxify the NT . Other biologically active sites of this protein (M, 130,000) are the antigrnic/ immunogenic epitopes . We are examining the structure of these active sites. Antigenically distinct NTs, called types A-G arc found in nature . The NT, when fully active, is a dichain protein made of an H and an L chain (M, N100,00 and 30,000, respectively) linked by at least one -S-S- bond. The paralysis of neuromuscular preparations (NMP, from mouse phrenic nerve-hemidiaphragm) induced by the dichain NT was antagonized if the NMPs were incubated with the isolated and purified H chain prior to or during incubation with the parrot
Second American Symposium
in the clinically positive pony, hematology and serum chemistry were normal in all ponies . Brain pathology was consistent with ELEM only in the clinically positive pony . Cytoplasmic changes (vacuolation, clumping) were observed in hepatocytes of all animals. Routine analysis of the cerebrospinal fluid (CSF) indicated chronic inflammation in both the clinically positive and one non~linical pony . Basic myelin protein of CSF was markedly elevated in the clinically positive pony as opposed to non~linical ponies (14 ng/ml vs . CL ng/ml). The results show susceptibility of ponies to Fusarium monilS%rme toxins) and suggest that basic myelin protein could be used as a marker in this condition. Additional studies involving strains isolated from the feed suspected in the North Carolina outbreak are in progress .
Fffert of T-2 mycotaxin on amino acid uptake in L~ myablastS. DAVID L. BUNNER, ELENA R. MORRIS, JUDITH G. PwcE and CHARLES F. MATSON (U .S . Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701, U .S .A .) . PROTEIN synthesis inhibition is a well documented direct cellular effect of T-2 mycotoxin. Other effects, such as alteration in amino acid uptake, have not been assessed . Since in vivo studies have documented hyperaminoacidemia after T-2 exposure and muscle is the largest single amino acid pool, amino acid uptake in L-6 myoblasts was measured . Confluent L-6 cells were studied after both S and 60 min exp~ure to T-2 and the cellular uptake (both acid-soluble and -precipitable) of aminoisobutyric acid, leucine and tyrosine were measured after a 1 and 3 min pulse of amino acid . Protein synthesis was reduced maximally (94- 9340) at higher doses of toxin (40 and 400 ng/ml) but was leas affected (SS - 8440 at the lowest dose (4 ng/ml) . Amino acid uptake was equally reduced at all doses (34- 7540). There was no significant difference between the S and 60 min exposure regarding protein synthesis inhibition or reduction in amino acid uptake . Radioisotopic scanning of thin-layer chromatography plates showed that leas than 340 of the parent T-2 was metabolized by 60 min. Amino acid metabolism was negligible. In conclusion, T-2 reduced both protein synthesis and amino acid uptake in L-6 myoblaata. Whether or not these two effects have any causal relationship could not be determined from the data .
Advaru+es in ncsaanh on jellyfish tanins and their pathogenic mechanisms. J~erx W. BURNETT and Gwev J. CALTON (Division of Dermatology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S .A .) . RESEARCx on the chemical characteristics of several jellyfish (Chrysaora quinquerirrha and Physolia physalis) toxins has been greatly aided by affinity immunochromatography procedures . The fishing tentacle of the sea nettle (Chrysaom quirrquecirrha) contains two polypeptides of mol. wt 190,000 and 100,000. The mesentery tentacles have a nematocyst venom containing three polypeptides of mol. wt 80,000, 160,000 and 110,000. A major action of these proteins is to increase monovalent and divalent cation transport across biological membranes. This transport increase is aided by the production of large holes in the membrane and can be reversed by the use of verapamil. Studies on the nematocyst venom of Portuguese man-0'war (Physaliaphysalis) have been aided by the use of an automated all sorter . With this technique, isolated nematocyst syspensions can be obtained free of all tentacle debris . This animal has two sizes of nematocysts, (10 and 23 Fart diameter), the smaller of which was found to be the more biologically active . The most prominent protein present in that nematocyst solution was a 70,000 mol.wt band . A aoas-reactivity to antigens present in either jellyfish venom has been demonstrated in monoclonal antibodies prepared from mice and polydonal antibodies from envenomatod patients . It has been found that the more seriously envenomated victims have higher circulating titers of species-specific polyclonal antibody . Additionally, patients envenonated by either species show considerable serological cross-reactivity with other jellyfish venous . A survey of envenomated patients has revealed several syndromes produced by wntact with these jellyfish. Persistent granulomatous lesions resulting from envenomation by Pelagic noctilurn have been accompanied by abnoruial natural killer (NK) cell activities aginst Chrysaora antigen for periods up to three years after resolution of the lesion . Patients with recurrent cutaneoua lesions following a single envenomation by Physalia and other jellyfish have been described. These individuals exhibit the expected serological response to the offending animal . In addition, one patient had an immunosuppression of her T cello evidenced by lymphokine blockage during one of her recurrent eruptions. Investigations on the effect of various blockers of inflammatory mediators on the increased cutaneous vasopermeability produced by various coelenterate venoms was undertaken. The results demonstrated that no one inhibitory drug was effective against the action exhibited by all venoms . This data demonstrates the fact that future therapy of jellyfish envenomation will be species-spaific. Tarantula venom: physirnl attributes and chromatogrtirphic jractlonation of Brachypelma emilia venom. C. D. CxwsrtwN, P. D. SwwRTZ, S. A. HUDIBURG, J. R. ScxwTZ and G. V. ODELL (Department of Biochemistry, Oklahoma State University, Oklahoma Agricultural Experiment Station, Stillwater, OK 74078, U .S .A .) .
Second American Symposium
13 7
TARANTULAS have become a popular exotic pet and the flashy orange and black body and grntle manner of Brachypelma emilia make it an attractive choice . The major hazard of this spider is its unicating hairs and although the venom is not deadly to man, this increased attention makes venom characterization important. Previous work on B. emilia venom includes comparison studies performed in this laboratory by Gabbiness et. ol. The venom has a pH of about 5.65, a solids content of 125 1+g/pl and an absorbance maximum of 26l nm . Protein content will be reported . Fractionation techniques will include gel filtration, polyacrylamide gel electrophoresis, thin-layer gel filtration and high performance liquid chromatography . Purification of the toxin is a major goal of this research and characteristics of this toxin will be reported . Hyaluronidase, protease and phospholipase activities of the venom will also be presented.
lsolotion and partial characterization of two lethal toxins from prairie rattlesnake (Crotalus viridis viridis) venom. TERRY R. COLBERG end CHARLOTTE L. OWNBY (Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, U .S .A). LYOPHILIZED prairie rattlesnake (Crotalus viridis viridis) venom puchased from biotoxins was fractionated by column chromatography . Two grams of the vrnom were dissolved in 0.01 M Tris buffer, pH 8.6, and after desalting by ultrafiltration was applied to a DEAF cellulose ion exchange column . The same buffer was used initially for elution until the absorbance at 280 nm returned to baseline . Then a NaCI gradirnt (0-0 .3 M) in the Tris buffer was used to elute the remaining protein. This procedure resulted in S fractions, all of which were tested for hemorrhagic, mytoxic and lethal activities by intramuscular injection into mice . Fraction Al from this rnlumn caused myonecrosis and hemorrhage at both 30 min and 24 hr after injection . It was pooled, concentrated, equilibrated with 0.01 M MOPS buffer, pH 7.2, and applied to an ultrogel AcA34 gel filtration column. Elution with this buffer yielded 8 fractions . Fraction BS from this column was non-hemorrhaic, myotoxic and lethal . This fraction was pooled, concrntrated and applied to a Whatman CM32 anion exchange column which was eluted with a linear salt gradient (0-0 .8 M NaCI) in 0.01 M MOPS buffer, pH 7.2 . This step resulted in 7 fractions of which Fractions C3 and CS caused death within 10 min after injection of 2.9 and 1.19 pg/g, respectively . Fraction C6 from this column was myotoxic but not lethal . Preliminary rraults indicate that Fraction C3 has an LDm (i.m .) of 0.03 Ng/g and Fraction CS of 0.09 F+8/g. These two toxins are being purified and assayed for protease, esterase and phospholipase activities .
Analysis ojjatty acids in the lipid rnmponents ojsnake venom. MARY E. CooKe, GEORGE V. ODELL, SuE A. HuD~auaG, JosE M. GUTIERREZ (Departmrnt of Biochemistry, Oklahoma State University, Oklahoma Agricultural Experiment Station, Stillwater, OK 74078, U.S .A ., and Inatituto Clodimiro Picado, Department of Biochemistry, San JosE, Costa Rita). THE LIPID fraction of Crotalus atrox lyophilized venom has been extracted. The methyl ester derivatives were analyzed by gas liquid chromatography on a Perkin Elmer 990 gas chromatograph . Several fatty acids were observed . Tentative identification by G.C. retention times indicates the presrnce of aprylic, esprit, lauric and palmitic acid . A significant amount of a higher molecular weight fatty acid has also been observed . Earlier studies of the Ngja ngja venom reported a large percentage of arachadonic acid . This suggests arachadonic as a possibility for the higher molecular weight acid . Further fractionation of the lipid portion of vrnom into neutral and phospholipid components and analysis on thin layer chromatographic plates is being performed. Also, a comparison of the lipid in adipose tissue, glands and venom of the snake will be conducted .
Botulinum neurotoxin : structurr-junction and similarity to tetanus. B. R. DASGupTA, S. BANDYOPADHYAY, A. W. CLARK, A. HERIAN, V. SATHYAMOORTHY and M. A. WOODY (FOOd Research InstitotC, University Of Wisconsin, Madison, WI 33706, U.S .A .) . SIMPSON proposed (Pharmac. Rev. 33, 133) that botulinum neurotoxin (NT) binds to cholinergic nerve terminals, then a part or the entire NT molecule is internalized and finally intracellular evrnt(s) block acetylcholine release. We conceive at least 2 active sites, 1 for binding and 1 for metabolic block; damage to either site would detoxify the NT . Other biologically active sites of this protein (M, 130,000) are the antigrnic/ immunogenic epitopes . We are examining the structure of these active sites. Antigenically distinct NTs, called types A-G arc found in nature . The NT, when fully active, is a dichain protein made of an H and an L chain (M, N100,00 and 30,000, respectively) linked by at least one -S-S- bond. The paralysis of neuromuscular preparations (NMP, from mouse phrenic nerve-hemidiaphragm) induced by the dichain NT was antagonized if the NMPs were incubated with the isolated and purified H chain prior to or during incubation with the parrot