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Microvascular changes in local haemorrhage produced experimentally by snake venoms
98
4th International Symposium on Animal, Plant and Microbial Toxins
The time course of 0-toxin production in a mixed culture of a 32 0- smR and b 300-2 OfrfR was quite similar to that of the parent strain PB6K . The hemolysin produced by the complementation was proved to be 0-toxinby the following experiments : (1) when the supernatant of a mixed culture was kept for 2months its hemolytic activity decreased from 128 to 2 units. The activity was fully restored by addition of M/200 cystein. (2) The hemolytic activity was completely inhibited by cholesterol. (3) 0-Antitoxin, devoid of a-antitoxin, neutralized the hemolytic activity completely . No genetic transfer could be demonstrated in the complementation . The complementation was detected through a membrane filter (Fuji-film microfilter No . 45) on blood agar, inoculating one of the two groups on the agar and another on the same spot of the membrane . A blood agar plate was streaked with b 300-11 0 frf R and coveredwith acellophane membrane on which a nutrient soft agar was inoculated with a 32 0-smR or PB6K. After incubation, complete hemolysis was found under the membrane and it was neutralized by 0-antitoxin. However, the reverse experiment (PB6K was omitted) gave no hemolysis. Trypsin, pronase and RNase, added to the soft agar together with group a or PB6K cells, were not effective in inhibiting the complementation. Of thirty heat-resistant strains isolated in outbreaks of food poisoning, twenty-eight strains were 0as described by Hoses et d. (1953) and belonging to group a, and two strains were 0+ but easily dissociated 0- mutants belonging to group a. All of twenty mutants of 0- group b isolated so far were gelatinase non-producers or leaky producers, and eighteen of 0- twenty-eight mutants of group a tested so far were gelatinase non-producers. When mutants of group a and group b were cross-streaked on Eiken Anaerobic Agar containing 3 percent gelatin, gelatinise activity was found around mixed colonies. The complementation in gelatinase production was also detected through a cellophane membrane just as the complementation in 0-toxin production, i.e . when group b mutants were grown on the agar and group a mutants on themembrane, gelatinase production was demonstrated. In addition, the group b mutants were incapable of hydrolysing sodium caseinate, fibrinogen, fibrin, hide powder, previously digested with activated papain and washed with polyvinylpyrrolidon according to KAMEYAMA and AKAMA (1970), and native bovine Achilles tendon, powdered together with dry ice. Some of group a mutants were also devoid of hydrolytic activity against these substrates. When complementation was examined, between the two groups, the hydrolysis of these substrates were demonstrated . Kappa antitoxin could neutralize the hydrolysis of hide powder treated as above by an enzyme (k-toxin) produced by complementation . HommA, J.
Y., ABE, C., OKADA, K., TANAMoTo, K. and HutAo, Y., Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan. THE BIOLOGICAL PROPERTIES OF THE PROTEIN MOIETY OF THE ENDOTOXIN OF PSEUDOMONAS AERUGINOSA
The protein fraction (OEP) of the endotoxin, derived from strain N 10 of Pseudomonas aeruginosa is markedly weaker than the lipopolysaccharide fraction (LPS) in Shwartzmian reaction, bone-marrow reaction and pyrogenic reaction, while stronger than LPS in IF inducing property and nonspecific property. It is noteworthy that the antitumor activity of OEP is remarkably stronger than that of LPS . Mice immunized with one of the two kinds of serologically different OEP can survive infection with each of the 14 different serotypes of Homma and the 7 different serotypes of Fisher. Using agar gel precipitin, OEP-HA tests, and the fluorescein antibody technique, it is found that OEP is a common antigen among these 21 serotype strains. Furthermore, OEP is also a common antigen among Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas aureofaciem (results of OEP-HA tests). HOM+A, M., Department
of Pathology, School of Medicine, Gunma University, Maebashi, Japan.
MICROVASCULAR CHANGES IN LOCAL HAEMORRHAGE PRODUCED EXPERIMENTALLY BY SNAKE VENOMS Local haemorrhage caused by crotaline snake envenomations starts shortly after the bite and lasts many hours. This study attempted to obtain ultrastructural information on microvascular changes due to haemorrhgic venoms . An electron microscopic examination of the microvessels of skeletal muscles, which were injured experimentally with Habu (7fimeresurusflavoviridis) and Mamushi (Agkistrodon balys) venom, revealed alterations indicative of bleeding from the capillaries and the venules. The changes consisted of endothelial gaps, which formed either through breakdown of endothelial cells or separation at the site of endothelial junction, and focal loss of the basement membrane. The lesions responsible for capillary and venule haemorrhage seem to be discontinuities of both the endothelium and the underlying basement membrane . TO%ICON 1975 Yd. 13
4th International Symposium on Animal, Plant and Microbial Toxins IBRAKm, S. A . and Khartoum, Sudan .
MASK,
99
A. R . M ., Department of Biochemistry, Faculty of Medicine, University of
ACTION OF PHOSPHOLIPASE A FROM BLACK MAMBA (DENDROASPIS POLYELEPIS) VENOM ON THE PHOSPHOLIPIDS OF HUMAN BLOOD Previous studies have shown that phospholipase A (phosphatide acyl hydrolase, EC 3 .1 .1 .4) contained in cobra (Neda ni#a) and sea snake (Enhydrina schislosa) venoms exhibited different substrate specificities when acting on the phospholipid components of human blood. The substrate specificity of the phospholipase A present in heat-treated black mamba venom, one of the most dangerous snakes in Africa, was studied using the phospholipids contained in human plasma, human red cell ghosts and intact erythrocytes as substrates. The study was conducted in a glycylglycine buffer system (0-1 M, pH 7 "3) . Black mamba venom enzyme was weakly active against the phospholipids of human plasma producing only 22 per cent hydrolysis. The activity was inhibited by sodium deoxycholate . The enzyme was completely inert towards the phospholipids of human red cell ghosts and intact erythrocytes . The results were confirmed chromatographically. The substrate specificities of black mamba venom phospholipase A are compared with those of cobra and sea-snake venoms enzymes . Clinically black mamba bite does not cause haemolysis and our results confirm this observation . INAYAMA, S ., ONKuRA, T., KAwAMATA, T., YANAGrrA, M .,1 ITAI, A. and IrrAKA, Y .,' (1) Pharmaceutical Laboratory, School of Medicine, Keio University, Shinjuku-ku, Tokyo, and (2) Faculty of Pharmaceutical Sciences, Tokyo University, Bunkyo-ku, Tokyo, Japan . AMBROSIC ACID, A NEW IRRITANT PRINCIPLE ISOLATED FROM
AMBROSIA ARTHEMISHFOLIA
In connection with our studies on the physiologically active substance in the genus of Gaillardia, Arthemisia and their relatives in Compositae, we have recently investigated a poisonous principle in the common rag weed, Ambrosia arthemisiifolia (Butakusa). Since this plant was introduced into Japan from North America almost one century ago, it has become widely distributed and notorious as a cause of pollen allergy (hay fever) and also as a poisonous weed in cattle feeds. In our investigation with collections of the plant in the environs of Tokyo, none of the known sesquiterpene lactones could be found, but a new modified pseudoguainolide named ambrosic acid was isolated, especially from the pollen . The fresh pollen collected from the flowers, air-dried chipped stems and leaves was submitted to individual percolation with hot chloroform . The acidic part of the extraction furnished the identical crystalline substance in a yield of 0-21, 0-08 and 0-02 per cent, respectively. C II 0 4 (M+ 264) ; m .p . 211-213°C ; (a)D + 77-0°C (CHCL 3) ;
vKB (cm 1) 3380,1732,1701,1622, 880 . To
R" H lb R = CHzCOC
Br(p)
FiG. 1 The structure of ambrosic acid was elucidated on the basis of the chemical and spectral data of I< and its derivatives. The absolute stereochemis~try of the molecule has been established by a X-ray crystallographic analysis of the p-bromophenacyl ester (Ib) as shown in Fig . l . Some results of a pharmacological investigation with ambrosic acid will be presented . INouE, K., IMAi, M ., KiTAGAwA, T. and NonmA, S ., Department of Chemistry, National Institute of Health,
Tokyo, Japan.
THE MECHANISM OF THE ACTION OF PRYMNESIUM TOXIN TOWARD MEMBRANES Prymnesin, a toxin produced by Prymnesium parvum is known to cause lysis of various erythrocytes and nucleated cells . The toxin produces damage to membranes causing leakage of intracellular constituents into medium . The mode of action of Prymnesin is not known, although the toxin was reported to behave as a surface-active agent. TOXICON 1975 Vol. 13
4th International Symposium on Animal, Plant and Microbial Toxins
The time course of 0-toxin production in a mixed culture of a 32 0- smR and b 300-2 OfrfR was quite similar to that of the parent strain PB6K . The hemolysin produced by the complementation was proved to be 0-toxinby the following experiments : (1) when the supernatant of a mixed culture was kept for 2months its hemolytic activity decreased from 128 to 2 units. The activity was fully restored by addition of M/200 cystein. (2) The hemolytic activity was completely inhibited by cholesterol. (3) 0-Antitoxin, devoid of a-antitoxin, neutralized the hemolytic activity completely . No genetic transfer could be demonstrated in the complementation . The complementation was detected through a membrane filter (Fuji-film microfilter No . 45) on blood agar, inoculating one of the two groups on the agar and another on the same spot of the membrane . A blood agar plate was streaked with b 300-11 0 frf R and coveredwith acellophane membrane on which a nutrient soft agar was inoculated with a 32 0-smR or PB6K. After incubation, complete hemolysis was found under the membrane and it was neutralized by 0-antitoxin. However, the reverse experiment (PB6K was omitted) gave no hemolysis. Trypsin, pronase and RNase, added to the soft agar together with group a or PB6K cells, were not effective in inhibiting the complementation. Of thirty heat-resistant strains isolated in outbreaks of food poisoning, twenty-eight strains were 0as described by Hoses et d. (1953) and belonging to group a, and two strains were 0+ but easily dissociated 0- mutants belonging to group a. All of twenty mutants of 0- group b isolated so far were gelatinase non-producers or leaky producers, and eighteen of 0- twenty-eight mutants of group a tested so far were gelatinase non-producers. When mutants of group a and group b were cross-streaked on Eiken Anaerobic Agar containing 3 percent gelatin, gelatinise activity was found around mixed colonies. The complementation in gelatinase production was also detected through a cellophane membrane just as the complementation in 0-toxin production, i.e . when group b mutants were grown on the agar and group a mutants on themembrane, gelatinase production was demonstrated. In addition, the group b mutants were incapable of hydrolysing sodium caseinate, fibrinogen, fibrin, hide powder, previously digested with activated papain and washed with polyvinylpyrrolidon according to KAMEYAMA and AKAMA (1970), and native bovine Achilles tendon, powdered together with dry ice. Some of group a mutants were also devoid of hydrolytic activity against these substrates. When complementation was examined, between the two groups, the hydrolysis of these substrates were demonstrated . Kappa antitoxin could neutralize the hydrolysis of hide powder treated as above by an enzyme (k-toxin) produced by complementation . HommA, J.
Y., ABE, C., OKADA, K., TANAMoTo, K. and HutAo, Y., Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan. THE BIOLOGICAL PROPERTIES OF THE PROTEIN MOIETY OF THE ENDOTOXIN OF PSEUDOMONAS AERUGINOSA
The protein fraction (OEP) of the endotoxin, derived from strain N 10 of Pseudomonas aeruginosa is markedly weaker than the lipopolysaccharide fraction (LPS) in Shwartzmian reaction, bone-marrow reaction and pyrogenic reaction, while stronger than LPS in IF inducing property and nonspecific property. It is noteworthy that the antitumor activity of OEP is remarkably stronger than that of LPS . Mice immunized with one of the two kinds of serologically different OEP can survive infection with each of the 14 different serotypes of Homma and the 7 different serotypes of Fisher. Using agar gel precipitin, OEP-HA tests, and the fluorescein antibody technique, it is found that OEP is a common antigen among these 21 serotype strains. Furthermore, OEP is also a common antigen among Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas aureofaciem (results of OEP-HA tests). HOM+A, M., Department
of Pathology, School of Medicine, Gunma University, Maebashi, Japan.
MICROVASCULAR CHANGES IN LOCAL HAEMORRHAGE PRODUCED EXPERIMENTALLY BY SNAKE VENOMS Local haemorrhage caused by crotaline snake envenomations starts shortly after the bite and lasts many hours. This study attempted to obtain ultrastructural information on microvascular changes due to haemorrhgic venoms . An electron microscopic examination of the microvessels of skeletal muscles, which were injured experimentally with Habu (7fimeresurusflavoviridis) and Mamushi (Agkistrodon balys) venom, revealed alterations indicative of bleeding from the capillaries and the venules. The changes consisted of endothelial gaps, which formed either through breakdown of endothelial cells or separation at the site of endothelial junction, and focal loss of the basement membrane. The lesions responsible for capillary and venule haemorrhage seem to be discontinuities of both the endothelium and the underlying basement membrane . TO%ICON 1975 Yd. 13
4th International Symposium on Animal, Plant and Microbial Toxins IBRAKm, S. A . and Khartoum, Sudan .
MASK,
99
A. R . M ., Department of Biochemistry, Faculty of Medicine, University of
ACTION OF PHOSPHOLIPASE A FROM BLACK MAMBA (DENDROASPIS POLYELEPIS) VENOM ON THE PHOSPHOLIPIDS OF HUMAN BLOOD Previous studies have shown that phospholipase A (phosphatide acyl hydrolase, EC 3 .1 .1 .4) contained in cobra (Neda ni#a) and sea snake (Enhydrina schislosa) venoms exhibited different substrate specificities when acting on the phospholipid components of human blood. The substrate specificity of the phospholipase A present in heat-treated black mamba venom, one of the most dangerous snakes in Africa, was studied using the phospholipids contained in human plasma, human red cell ghosts and intact erythrocytes as substrates. The study was conducted in a glycylglycine buffer system (0-1 M, pH 7 "3) . Black mamba venom enzyme was weakly active against the phospholipids of human plasma producing only 22 per cent hydrolysis. The activity was inhibited by sodium deoxycholate . The enzyme was completely inert towards the phospholipids of human red cell ghosts and intact erythrocytes . The results were confirmed chromatographically. The substrate specificities of black mamba venom phospholipase A are compared with those of cobra and sea-snake venoms enzymes . Clinically black mamba bite does not cause haemolysis and our results confirm this observation . INAYAMA, S ., ONKuRA, T., KAwAMATA, T., YANAGrrA, M .,1 ITAI, A. and IrrAKA, Y .,' (1) Pharmaceutical Laboratory, School of Medicine, Keio University, Shinjuku-ku, Tokyo, and (2) Faculty of Pharmaceutical Sciences, Tokyo University, Bunkyo-ku, Tokyo, Japan . AMBROSIC ACID, A NEW IRRITANT PRINCIPLE ISOLATED FROM
AMBROSIA ARTHEMISHFOLIA
In connection with our studies on the physiologically active substance in the genus of Gaillardia, Arthemisia and their relatives in Compositae, we have recently investigated a poisonous principle in the common rag weed, Ambrosia arthemisiifolia (Butakusa). Since this plant was introduced into Japan from North America almost one century ago, it has become widely distributed and notorious as a cause of pollen allergy (hay fever) and also as a poisonous weed in cattle feeds. In our investigation with collections of the plant in the environs of Tokyo, none of the known sesquiterpene lactones could be found, but a new modified pseudoguainolide named ambrosic acid was isolated, especially from the pollen . The fresh pollen collected from the flowers, air-dried chipped stems and leaves was submitted to individual percolation with hot chloroform . The acidic part of the extraction furnished the identical crystalline substance in a yield of 0-21, 0-08 and 0-02 per cent, respectively. C II 0 4 (M+ 264) ; m .p . 211-213°C ; (a)D + 77-0°C (CHCL 3) ;
vKB (cm 1) 3380,1732,1701,1622, 880 . To
R" H lb R = CHzCOC
Br(p)
FiG. 1 The structure of ambrosic acid was elucidated on the basis of the chemical and spectral data of I< and its derivatives. The absolute stereochemis~try of the molecule has been established by a X-ray crystallographic analysis of the p-bromophenacyl ester (Ib) as shown in Fig . l . Some results of a pharmacological investigation with ambrosic acid will be presented . INouE, K., IMAi, M ., KiTAGAwA, T. and NonmA, S ., Department of Chemistry, National Institute of Health,
Tokyo, Japan.
THE MECHANISM OF THE ACTION OF PRYMNESIUM TOXIN TOWARD MEMBRANES Prymnesin, a toxin produced by Prymnesium parvum is known to cause lysis of various erythrocytes and nucleated cells . The toxin produces damage to membranes causing leakage of intracellular constituents into medium . The mode of action of Prymnesin is not known, although the toxin was reported to behave as a surface-active agent. TOXICON 1975 Vol. 13