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An apparently new toxin from coral reef crabs
30
First A s i a - Pacific Congress
Toxins and fungi. G. LIM (Botany Department, National University of Singapore, Singapore). MUSHROOM toxins are considered in 5 categories: gastric, cellular, nerve, hallucinogenic and alcohol reaction. The species of mushrooms implicated in such poisonings are named and some reference made to local examples. Toxins from moulds are also dealt with. The different kinds of toxigenic microfungi and the toxins produced are described. The mycotoxicoses presented include ergotism, aflatoxicosis, clavacitoxicosis, islanditoxicosis and fusariotoxicosis. The species of microfungi occurring locally that may be involved in such toxicoses are mentioned. Respiratory failure and its non-antivenin treatment in 37 adult neurtoxic snake-bite patients. S. LIMTHONGKUL, C. POCHNUGOOL and K. MEEMANO (Department of Medicine, Chulalongkorn Hospital, Faculty of Medicine, Chulalongkorn University, Bangkok 10500, Thailand). To INCREASEunderstanding about the natural history of respiratory failure and its non-antivenin treatment in adult neurotoxic snake-bite patients, 37 adult patients bitten by neurotoxic snakes were studied. Out of 37 nonantivenin treated patients, 32 developed respiratory muscle fatigue which required assisted ventilation. All the patients survived and did well after being weaned from the respirator. The average onset of muscle fatigue and the duration of assisted ventilation were 5.13 h (0.5 - 15.5 h) and 44.5 h ( 8 - 143.5 h), respectively. There were significant inverse correlations between the start of assisted ventilation and the distance between the two fang marks (y = 1.6864- 0.065x; P<0.05) and the duration of assisted ventilation (y = 64.9211 -3.9773x; P<0.001). Unresponsiveness during assisted ventilation was due to muscle paralysis rather than unconsciousness. Assisted ventilation and respiratory supportive care are most important in the management of neurotoxic snake-bite patients. Such patients Should be intubated with an orotracheal tube when upper respiratory muscle paralysis develops and should be followed by assisted ventilation as soon as there is evidence of abdominal paradox and/ or respiratory alternans. Ultrastructure o f choroid plexus and epiplexus cells in cat following a cisternal injection o f crotoxin complex. E . A . LING, P. GOPALAKRISHNAKONE and C. K. TAN (Department of Anatomy, National University of Singapore, Kent Ridge, Singapore 0511). EIGHT cats weighing between 2.5 and 3.5 kg were used. Under sodium pentobarbitone anaesthesia, 0.2 ml of phospholipase A2 (crotoxin complex, 25 /~g per kg body weight) was injected into the cisterna magna. For controls, two animals were injected with normal saline. All animals were sacrificed by vascular perfusion with a mixture of 2o/0 paraformaldehyde and 3% glutaraldehyde in cacodylate buffer, pH 7.4, at 1, 2 and 5 h after injection. The choroid plexus of the 4th ventricle was processed for scanning and transmission electron microscopy (SEM, TEM). SEM of the ventricular surface of the choroid plexus of the control animals showed closely packed, dome-shaped epithelial cells. Resting on them were numerous pleomorphic epiplexus cells, the majority of which were stellate-shaped with four to eight radiating cytoplasmic processes. After the injection of phospholipase the epiplexus cells showed spreading of their cytoplasmic processes forming a thin membranous canopy over the epithelial cells. In areas not covered by the cytoplasmic expansions, each epithelial cell showed 1 - 5 blebs, some o f which appeared to have ruptured. TEM showed that the blebs were cytoplasmic protrusions containing amorphous material with free- and polyribosomes. Portions of cytoplasm bearing similar features to the blebs were often seen in the ventricular lumen, being phagocytosed by the overlying epiplexus cells. The results suggest that phospholipase A can induce an apocrine type of secretion of choroid epithelial cells. The alteration of the surface morphology of epiplexus cells is a reflection of their functional activities. The phagocytosis of the secretOry products from the epithelial cells by the epiplexus cells suggests a close functional relationship between the two cell types.
A n apparently new toxin from coral reef crabs. L. E. LLEWELLYN and R. ENDEAN (Department of Zoology, University of Queensland, St. Lucia, Brisbane 4067, Australia). A WATERSOLUBLEtoxin, chromatographically and pharmacologically distinct from saxitoxin, tetrodotoxin and palytoxin, has been found in coral reef crabs. The toxin was extracted from specimens of Atergatis floridus Lophozozymous pictor, Eriphia sebana and Carpilius convexus (F. Xanthidae), Grapsus albolineatus (F. Grapsidae) and Daldorfia horrida (F. Parthenopidae). The LDs0of the partially purified toxin after i.p. injection into 20 g male mice was 180 mg/kg. Low doses of the toxin produced the following signs in mice: blanching, piloerection, d i a r r h ~ a , tremors, laboured respiration, inactivity, loss of body temperature and flaccid paralysis. Respiratory arrest preceded cardiac arrest, which occurred within 72 h after administration of the toxin. High doses of the toxin caused a protracted tonic spasm. Death occurred between 8 and 15 rain after administration of
First Asia - Pacific Congress
31
the toxin. The toxin caused a sustained contraction of isolated guinea-pig ileal strips which was unaffected by tetrodotoxin, but blocked immediately by atropine. Negative inotropy and negative chronotropy in the spontaneously beating right atrium and negative inotropy in the electrically driven left atrium of guinea-pigs was produced by the toxin. The toxin had no direct effect on the guinea-pig vas deferens but potentiated the KC1induced contraction of this preparation. The response of the rat phrenic nerve - hemidiaphragm preparation to indirect stimulation was blocked and the response of the preparation to direct stimulation was reduced. Resting muscle tension was decreased slightly. It is suggested that this toxin acts either by causing the release of acetylcholine or by a direct action on acetylchuline receptors. This toxin does not appear to have been found previously in coral reef crabs.
The use o f ultrasonography in the treatment o f habu bites (Trimeresurus fiavoviridis envenonmation). M. MA~,IRO (Department of Surgery, Okinawa Prefectural Chubu Hospital, Gushikawa City, Okinawa, Japan 904 - 22). REALTIME B-mode ultrasonography was used to assess the pathologic changes of local tissue after habu bites in selected cases. The purpose of the study was to investigate the clinical value of ultrasonography, which is noninvasive and can be readily repeated at the bed-side, in improving the treatment of habu bites. Ultrasonographic examination was performed on 8 severe cases out of a total of 46 cases treated at Okinawa Prefectural Chubu Hospital during 1986. The uninvolved extremity served as the control.,Ultrasonography dearly distinguished the swelling of skin, subcutaneous tissue and muscle layers and showed the depth of pathologic changes, whether or not it reached the subfascial plane. Progression of the pathologic changes could also be detected by increased echogcnicity and the degree of swelling itself could be measured objectively. It is concluded that ultrasonography is a useful tool in the treatment of habu bites. It is especially useful in severe cases, by demonstrating the involvement of muscular tissue and the need for increasing the dose of antivcnom. It is also helpful in deciding on the indications for decompression faciotomy to salvage limb function by preventing massive muscle necrosis from increased compartment pressure. The final decision to perform faciotomy should be made by tissue pressure measurement, however.
Ecchymosia as a clinical predictor in green pit viper bite. S. MAI-IASA~DANA,S. RATANANDAand B. AKKAWAT (Department of Medicine, Chulalongkorn Hospital Medical School, Bangkok 10500, Thailand). The purpose of the study was to relate the ecchymosis with the clinical severity of green pit viper bite. By using the platelet numbers as a gold standard and the ecchymosis as a test, the patterns of 65 green pit viper victims were classified into mild and serious cases. In mild cases the platelet count was more than 150 × 109/1 and in serious cases the value was less than 150 × 109/1. The only significant clinical finding among the 36 'mild' cases was edema. The 29 serious cases could be divided into 2 subgroups: (a) the 'severe' cases were those who developed systemic bleeding; (b) the 'moderate' those who did not. Gingival bleeding was the most common manifestation. Ecchymosis devdoped in 24 out of the 65 cases. The ecchymoses were noted in all 13 severe cases and in 11 of the 16 moderate cases. The sensitivity and the specificity of the ecchymosis in predicting the severity of the bite were 82.8°70 and 100070, respectively. Ecchymosis can be a good predictor for clinical classification of severity when the platelet count is not available. Systemic bleeding is a definite clinical sign in severe cases.
The purification and characterization o f the prothrombin activator o f the venom o f Pseudonaja textilis. P. P. MASCI,' A. N. WH1TAKERs and J. DE JERSEY2 (~Department of Medicine, University of Queensland, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane 4102, and 2Department of Biochemistry, University of Queensland, St. Lucia 4067, Australia). THE VENOM of the Australian snake Pseudonaja textilis contains a prothrombin activator which converts prothrombin to a-thrombin in the absence of factor V, factor X, calcium and phospholipid. Purified prothrombin is first converted to an intermediate fragment of molecular weight 57,000. The prothrombin activator of the venom of P. textilis can be purified by chromatography on con A - S e p h a r o s e 4B and gel filtration on Sephacryl S-300. The prothrombin activator is a single band on PAGE at pH 8.6, and on SDSPAGE in the absence of/~-mercaptoethanol four bands are observed with molecular weights of 190,000, 170,000, 65,000 and 60,000. Purified prothrombin activator clots citrated plasma in the presence and absence of calcium. Cross-linked chains are produced, indicating that plasma factor XIII is activated. Aluminium hydroxide absorbed plasmas from patients on warfarin is clotted by P. textilis prothrombin activator, suggesting that descarboxyprothrombin is activated to thrombin. The prothrombin activator hydrolyses the synthetic substrate S-2222 (Kin = 0.04 mM), as well as a number of other peptide substrates. Antisera raised to the purified prothrombin activator of the venom of P. textilia have been shown to give a single immunoprecipitin
First A s i a - Pacific Congress
Toxins and fungi. G. LIM (Botany Department, National University of Singapore, Singapore). MUSHROOM toxins are considered in 5 categories: gastric, cellular, nerve, hallucinogenic and alcohol reaction. The species of mushrooms implicated in such poisonings are named and some reference made to local examples. Toxins from moulds are also dealt with. The different kinds of toxigenic microfungi and the toxins produced are described. The mycotoxicoses presented include ergotism, aflatoxicosis, clavacitoxicosis, islanditoxicosis and fusariotoxicosis. The species of microfungi occurring locally that may be involved in such toxicoses are mentioned. Respiratory failure and its non-antivenin treatment in 37 adult neurtoxic snake-bite patients. S. LIMTHONGKUL, C. POCHNUGOOL and K. MEEMANO (Department of Medicine, Chulalongkorn Hospital, Faculty of Medicine, Chulalongkorn University, Bangkok 10500, Thailand). To INCREASEunderstanding about the natural history of respiratory failure and its non-antivenin treatment in adult neurotoxic snake-bite patients, 37 adult patients bitten by neurotoxic snakes were studied. Out of 37 nonantivenin treated patients, 32 developed respiratory muscle fatigue which required assisted ventilation. All the patients survived and did well after being weaned from the respirator. The average onset of muscle fatigue and the duration of assisted ventilation were 5.13 h (0.5 - 15.5 h) and 44.5 h ( 8 - 143.5 h), respectively. There were significant inverse correlations between the start of assisted ventilation and the distance between the two fang marks (y = 1.6864- 0.065x; P<0.05) and the duration of assisted ventilation (y = 64.9211 -3.9773x; P<0.001). Unresponsiveness during assisted ventilation was due to muscle paralysis rather than unconsciousness. Assisted ventilation and respiratory supportive care are most important in the management of neurotoxic snake-bite patients. Such patients Should be intubated with an orotracheal tube when upper respiratory muscle paralysis develops and should be followed by assisted ventilation as soon as there is evidence of abdominal paradox and/ or respiratory alternans. Ultrastructure o f choroid plexus and epiplexus cells in cat following a cisternal injection o f crotoxin complex. E . A . LING, P. GOPALAKRISHNAKONE and C. K. TAN (Department of Anatomy, National University of Singapore, Kent Ridge, Singapore 0511). EIGHT cats weighing between 2.5 and 3.5 kg were used. Under sodium pentobarbitone anaesthesia, 0.2 ml of phospholipase A2 (crotoxin complex, 25 /~g per kg body weight) was injected into the cisterna magna. For controls, two animals were injected with normal saline. All animals were sacrificed by vascular perfusion with a mixture of 2o/0 paraformaldehyde and 3% glutaraldehyde in cacodylate buffer, pH 7.4, at 1, 2 and 5 h after injection. The choroid plexus of the 4th ventricle was processed for scanning and transmission electron microscopy (SEM, TEM). SEM of the ventricular surface of the choroid plexus of the control animals showed closely packed, dome-shaped epithelial cells. Resting on them were numerous pleomorphic epiplexus cells, the majority of which were stellate-shaped with four to eight radiating cytoplasmic processes. After the injection of phospholipase the epiplexus cells showed spreading of their cytoplasmic processes forming a thin membranous canopy over the epithelial cells. In areas not covered by the cytoplasmic expansions, each epithelial cell showed 1 - 5 blebs, some o f which appeared to have ruptured. TEM showed that the blebs were cytoplasmic protrusions containing amorphous material with free- and polyribosomes. Portions of cytoplasm bearing similar features to the blebs were often seen in the ventricular lumen, being phagocytosed by the overlying epiplexus cells. The results suggest that phospholipase A can induce an apocrine type of secretion of choroid epithelial cells. The alteration of the surface morphology of epiplexus cells is a reflection of their functional activities. The phagocytosis of the secretOry products from the epithelial cells by the epiplexus cells suggests a close functional relationship between the two cell types.
A n apparently new toxin from coral reef crabs. L. E. LLEWELLYN and R. ENDEAN (Department of Zoology, University of Queensland, St. Lucia, Brisbane 4067, Australia). A WATERSOLUBLEtoxin, chromatographically and pharmacologically distinct from saxitoxin, tetrodotoxin and palytoxin, has been found in coral reef crabs. The toxin was extracted from specimens of Atergatis floridus Lophozozymous pictor, Eriphia sebana and Carpilius convexus (F. Xanthidae), Grapsus albolineatus (F. Grapsidae) and Daldorfia horrida (F. Parthenopidae). The LDs0of the partially purified toxin after i.p. injection into 20 g male mice was 180 mg/kg. Low doses of the toxin produced the following signs in mice: blanching, piloerection, d i a r r h ~ a , tremors, laboured respiration, inactivity, loss of body temperature and flaccid paralysis. Respiratory arrest preceded cardiac arrest, which occurred within 72 h after administration of the toxin. High doses of the toxin caused a protracted tonic spasm. Death occurred between 8 and 15 rain after administration of
First Asia - Pacific Congress
31
the toxin. The toxin caused a sustained contraction of isolated guinea-pig ileal strips which was unaffected by tetrodotoxin, but blocked immediately by atropine. Negative inotropy and negative chronotropy in the spontaneously beating right atrium and negative inotropy in the electrically driven left atrium of guinea-pigs was produced by the toxin. The toxin had no direct effect on the guinea-pig vas deferens but potentiated the KC1induced contraction of this preparation. The response of the rat phrenic nerve - hemidiaphragm preparation to indirect stimulation was blocked and the response of the preparation to direct stimulation was reduced. Resting muscle tension was decreased slightly. It is suggested that this toxin acts either by causing the release of acetylcholine or by a direct action on acetylchuline receptors. This toxin does not appear to have been found previously in coral reef crabs.
The use o f ultrasonography in the treatment o f habu bites (Trimeresurus fiavoviridis envenonmation). M. MA~,IRO (Department of Surgery, Okinawa Prefectural Chubu Hospital, Gushikawa City, Okinawa, Japan 904 - 22). REALTIME B-mode ultrasonography was used to assess the pathologic changes of local tissue after habu bites in selected cases. The purpose of the study was to investigate the clinical value of ultrasonography, which is noninvasive and can be readily repeated at the bed-side, in improving the treatment of habu bites. Ultrasonographic examination was performed on 8 severe cases out of a total of 46 cases treated at Okinawa Prefectural Chubu Hospital during 1986. The uninvolved extremity served as the control.,Ultrasonography dearly distinguished the swelling of skin, subcutaneous tissue and muscle layers and showed the depth of pathologic changes, whether or not it reached the subfascial plane. Progression of the pathologic changes could also be detected by increased echogcnicity and the degree of swelling itself could be measured objectively. It is concluded that ultrasonography is a useful tool in the treatment of habu bites. It is especially useful in severe cases, by demonstrating the involvement of muscular tissue and the need for increasing the dose of antivcnom. It is also helpful in deciding on the indications for decompression faciotomy to salvage limb function by preventing massive muscle necrosis from increased compartment pressure. The final decision to perform faciotomy should be made by tissue pressure measurement, however.
Ecchymosia as a clinical predictor in green pit viper bite. S. MAI-IASA~DANA,S. RATANANDAand B. AKKAWAT (Department of Medicine, Chulalongkorn Hospital Medical School, Bangkok 10500, Thailand). The purpose of the study was to relate the ecchymosis with the clinical severity of green pit viper bite. By using the platelet numbers as a gold standard and the ecchymosis as a test, the patterns of 65 green pit viper victims were classified into mild and serious cases. In mild cases the platelet count was more than 150 × 109/1 and in serious cases the value was less than 150 × 109/1. The only significant clinical finding among the 36 'mild' cases was edema. The 29 serious cases could be divided into 2 subgroups: (a) the 'severe' cases were those who developed systemic bleeding; (b) the 'moderate' those who did not. Gingival bleeding was the most common manifestation. Ecchymosis devdoped in 24 out of the 65 cases. The ecchymoses were noted in all 13 severe cases and in 11 of the 16 moderate cases. The sensitivity and the specificity of the ecchymosis in predicting the severity of the bite were 82.8°70 and 100070, respectively. Ecchymosis can be a good predictor for clinical classification of severity when the platelet count is not available. Systemic bleeding is a definite clinical sign in severe cases.
The purification and characterization o f the prothrombin activator o f the venom o f Pseudonaja textilis. P. P. MASCI,' A. N. WH1TAKERs and J. DE JERSEY2 (~Department of Medicine, University of Queensland, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane 4102, and 2Department of Biochemistry, University of Queensland, St. Lucia 4067, Australia). THE VENOM of the Australian snake Pseudonaja textilis contains a prothrombin activator which converts prothrombin to a-thrombin in the absence of factor V, factor X, calcium and phospholipid. Purified prothrombin is first converted to an intermediate fragment of molecular weight 57,000. The prothrombin activator of the venom of P. textilis can be purified by chromatography on con A - S e p h a r o s e 4B and gel filtration on Sephacryl S-300. The prothrombin activator is a single band on PAGE at pH 8.6, and on SDSPAGE in the absence of/~-mercaptoethanol four bands are observed with molecular weights of 190,000, 170,000, 65,000 and 60,000. Purified prothrombin activator clots citrated plasma in the presence and absence of calcium. Cross-linked chains are produced, indicating that plasma factor XIII is activated. Aluminium hydroxide absorbed plasmas from patients on warfarin is clotted by P. textilis prothrombin activator, suggesting that descarboxyprothrombin is activated to thrombin. The prothrombin activator hydrolyses the synthetic substrate S-2222 (Kin = 0.04 mM), as well as a number of other peptide substrates. Antisera raised to the purified prothrombin activator of the venom of P. textilia have been shown to give a single immunoprecipitin