Abstracts from Other Presentations at the International Symposium on Toxins and Natural Products in Honor of Professor Anthony T. Tu

Abstract

Investigation of the Structure, Dynamics, and Folding of Snake Venom Proteins Chin Yu* and T. K. C. Kumar Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan Cardiotoxins isolated from Taiwan cobra venom (Naja naja atra) are small molecular weight proteins (∼ 7 kDa), containing four disulfide bonds. To‐date, different cardiotoxin isoforms (CTXI, CTXII, CTX III, CTXIV, and CTXV) have been isolated from the venom of Naja naja atra. The three‐dimensional structures of all the five cardiotoxin isoforms have been solved by multidimensional NMR techniques. Critical comparison of the structures of cardiotoxins reveal a common structural feature responsible for the lethal activity of cardiotoxins. Although the cardiotoxins show very high structural homology they exhibit significant differences in their lethal potencies. The observed differences in the lethal potencies are found to depend on the degree of exposure of the positive charge of an invariant lysine. Backbone dynamics of CTXIII has been studied by carbon‐13 relaxation measurements at natural abundance. The overall rotational correlation co‐efficient of the protein has been estimated to be 4.8 ns. Most of the residues in CTXIII have been observed to exhibit fast (τe < 30 ps) restricted motions (S2 = 0.79–0.89). The functional important residues located at the tips of three lops are relatively flexible. The structural stability of CTXIII had been probed by hydrogen–deuterium exchange monitored by NMR spectroscopy. Among the five beta strands in the toxin, beta strand III is found to constitute the stability core. The stability of the triple stranded beta‐sheet domain is markedly higher than that of the double stranded beta‐sheet domain. The refolding of CTXIII monitored by a variety of biophysical techniques reveals that the toxin refolds completely within a time span of 200 milliseconds. The chronology of the folding events in CTXIII monitored by quenched‐flow H/D exchange shows that the triple‐stranded beta‐sheet domain folds faster than the double stranded beta‐sheet domain. These results will be extensively discussed. Occurrence of GM4 Ganglioside as the Major Glycosphingolipid in Shark Liver Yu‐Teh Li* and Su‐Chen Li Department of Biochemistry, Tulane University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112, USA Glycosphingolipids (GSLs) occur in all eukaryotic cells. Each GSL contains a carbohydrate‐head group covalently linked to a lipophilic ceramide tail, which anchors the molecule to the cell membrane. GSLs and their derivatives have been shown to mediate cell adhesion, signalling, receptor modulation, apoptosis, growth and differentation. Over three hundred different GSLs have been isolated from various sources. Compared with GSLs from tissues of higher animals, GSLs in the tissues of marine organisms are not well studied. We found that GSLs in tissues of marine organisms are quite tissue specific. For example, GM2 is the major ganglioside found in mullet roe (Li et al., ), whereas GM3 is the major ganglioside found in mullet milt (Zhu et al., ). Sharks are known to have large livers storing large amounts of lipid materials that have been shown to be responsible for maintaining their buoyancy. While studying the distribution of GSLs in marine organisms, we found that the liver samples from Odontaspis taurus, Negaprion brevirostris, Sphyrna lewini, Mustelus griseus, Mustelus manazo and Prionaceglauca were unusually rich in ganglioside GM4. In man, GM4 is associated with myelin. Immunohistochemical analysis using a monoclonal antibody against GM4 revealed that the hepatocytes of white shark (M. griseus) was filled with lipid droplets and that GM4 was primarily associated with the limiting membranes, sequestering the lipid droplets. We have performed a detailed analysis of the GSLs in the liver of sand shark (O. taurus) and found that it contained approximately 110 nmol of lipid‐bound sialic acid per g wet tissue of which 80% was GM4. Using chloroform/methanol extraction and chromatographic separation of GSLs by DEAE‐Sephadex A‐25 and Iatrobeads columns, we have isolated GM4 from sand shark liver in pure form with a yield of approximately 5 mg per 100 g wet tissue. The structures of both the sugar chain and the ceramide moiety of this GM4 were analyzed by chemical analysis, mass spectrometry, and NMR spectroscopy. The Antioxidant Properties and Xanthine Oxidase Inhibitory Activities of Compounds Isolated from Marine Organisms Christopher H.K. Cheng1,* and Tatsuo Higa2 1Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong 2Department of Chemistry, College of Science, University of the Ryukyus, Nishihara, Okinawa 903‐0213, Japan Aerobic organisms depend on the supply of oxygen for their life processes. However, oxygen is also potentially toxic. One aspect of oxygen toxicity is the formation of reactive oxygen species that can attack cellular components such as proteins, lipids and DNA causing all kinds of damages including protein inactivation, lipid peroxidation and gene mutations. Various cellular defense mechanisms are available to protect the cells from the damaging effects of such reactive oxygen species including free radical scavenging enzymes and the small molecule antioxidants such as ascorbic acid, α‐tocopherol and glutathione. On the other hand, cellular production of free radicals is also physiologically important. The production of free radicals in the macrophages to kill the engulfed microorganisms is one example. Thus a carefully controlled extent of free radical production compatible with life is essential for the normal operation of a living organism. Pathological conditions will thus arise if the balance is disturbed, either by an increased production of free radicals, or by an attenuation of the defence mechanisms. Exogenously introduced antioxidants might find therapeutic applications under such situations. In this paper the antioxidant properties of a number of compounds isolated from marine organisms will be investigated, namely 2,3‐dithiomethyl‐4,6‐dibromoindole, 10‐isothiocyanato‐4‐amorphene, 1‐isothiocyanato‐aromadendrane, 2′‐demethylaplysinopsin, strongylophorine‐3, tetraprenylhydroquinone, laurinterol, and 2‐hydroxy‐2′,4,4′,6‐tetrabromodiphenyl ether. The antioxidant properties were investigated in rat erythrocytes exposed to 2,2′‐azobis(2‐amidinopropane) dihydrochloride. Among the compounds investigated, tetrabromodiphenyl ether also exhibits potent inhibitory activities against the enzyme xanthine oxidase. This enzyme is involved in the hypoxia‐reperfusion damage of tissues where free radicals play a role. The significance of these findings will be discussed. Mushroom Poisonings in Japan Kunio Gonmori Department of Forensic Medicine, Akita University School of Medicine, Akita 010‐8543, Japan; E‐mail: gonmori@med.akita-u.ac.jp It is estimated that there are over 5,000 species of mushrooms all over the world of which about 300 species are edible. Only 20–25% of mushrooms have been named, and 3% of these are poisonous. During the 24 years from 1974 to 1997, 1,057 cases of mushroom poisoning have been reported to the Ministry of Health and Welfare through the Regional Health Center. Almost half of these cases were caused by Rhodophyllus rhodopolius and Lampes japonicusteromyc. These mushrooms are well known as poisonous mushrooms in Japan. Fortunately, most of these exposures were less serious and required very little medical treatment. Moreover, cases of poisoning by the most dangerous mushroom, the Amanita species, are rare. Amanita virosa is one of the most dangerous mushrooms. The color is white and feels soft to touch. This mushroom is called “the destroying angel.” The Amanita virosa is usually found alone or in a small, scattered group and the young, unexpanded caps resemble edible agaricus mushrooms. Symptoms of poisoning by Amanita virosa are like the deadly Amanita phalloides, and include vomiting, diarrhea, and cramps; kidney and liver dysfunction follow and if treatment is not available can even result in death. Rhodophyllus rhodopolius is well known as a poisonous mushroom in Japan. However, amateur mushroom hunters often mistake this one as an edible mushroom because of its similar shape and color. This group has caused almost 30% of all poisoning cases. Lampteromyces japonicus. The cap's shape is semicircular or horse bean, eccentric, asymmetrical, shell‐ or spatula shaped. Lampteromyces japonicus grows gregariously on dead beech and maple‐trunks in the woods. This mushroom has caused almost 26% of all poisoning cases. “Magic mushroom” is the general term of the mushrooms containing hallucinogens, such as psilocybin and psilocin. Although the components, psilocybin and psilocin, have been regulated by law as narcotic drugs in Japan, its cultivation and possession was not banned by law. However, the mushroom which contains psilocybin and/or psilocin has been designated as a narcotic plant by law as of this June and hence, its cultivation as well as possession is now illegal. A fatal case of magic mushroom poisoning was analyzed. A 27 years old man was found in an irrigation canal. Two cultivating pots of mushroom were found in his room. The culture pots were put on the cultivation equipment of our laboratory, and the mushrooms were grown two weeks later. The mushroom was identified as Psilocybe subcubensis from the spore. Hallucinogenic substances from the body fluids and cultivated mushrooms were detected. Syntheses and Paralytic Activities of Squaryl Amino Acid‐Containing Polyamine Toxins Tetsuro Shinada,1 Yoshito Nakagawa,1 Ken‐ichi Hayashi,1 Gerald Corzo,2 Terumi Nakajima,2 and Yasufumi Ohfune2 1Graduate School of Science, Osaka City University, Osaka 558‐8585, Japan 2Suntory Institute for Bioorganic Research, Mishima‐gun, Osaka 618‐8503, Japan Philanthotoxin (PhTX) from Philantus triangulum and nephilatoxins (NPTX) from Nephila cl