Journal of natural toxins最新文献

The aim of this study was to obtain anti-snake antiserum by optimizing the conditions of extraction and purification and test its ability to neutralize local myonecrosis. Extraction and purification was achieved through adjustment of the pH, pepsin concentration, time of digestion, and caprylic acid concentration. Our results indicate that the best conditions to obtain anti-snake antiserum from ammonium sulfate fractionated plasma are pH 3.3, 3.5 g/l pepsin, digestion for 90 min at 37 degrees C, and 0.5% caprylic acid. Antiserum purified using this method has greater neutralizing ability of myonecrosis than ammonium sulfate (ammSO4) fractionated product.

Between October 30 and November 4, 2000, eleven persons were intoxicated due to ingestion of a serranid fish Epinephelus sp. in Kochi Prefecture, Japan. Their symptoms were mainly featured by severe muscle pain, low back pain, and discharge of black urine. Serum creatine phosphokinase (CPK) levels of victims were higher (700-23,800 IU/l) than normal values, and their recovery times were more than one month. Immediately after the incident, the leftovers were collected for investigation. The causative agent was identified as palytoxin (PTX) on the basis of delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). To our knowledge, this is the first report on palytoxin poisoning with serranid fish.

A novel fibrinogenolytic protease, named alpha-mucrofibrase, was purified from the venom of Chinese Habu (Trimeresurus mucrosquamatus) by DEAE-Sephadex A-50 ion-exchange chromatography and Sephadex G-100 (super fine) gel filtration alpha-Mucrofibrase is a single-chain polypeptide of approximately 29 kDa. It is stable even at 95 degrees C, and the most susceptible hydrolysis substrate is S-2302. It cleaved primarily the Aalpha chain of fibrinogen followed by the Bbeta chain, while the gamma chain was partially affected. N-terminal sequence of this fibrinogenolytic enzyme has great homology with those of other snake venom serine proteases. The esterase activity of alpha-mucrofibrase is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by metal chelator (EDTA), suggesting this fibrinogenase belongs to the venom serine protease family.

Sea snake venoms contain less protein than those of land snakes (Toom et al., 1969). Sea snake venoms lack arginine ester hydrolyzing activity, whereas those of Crotalidae and Viperidae have such activity (Tu et al., 1966). Sea snakes live in salty water, and their venoms may be different from those of land snakes. Because of the difficulty in obtaining sea snake venoms, information about sea snake venoms is quite incomplete. NGF is commonly present in the venoms of land snakes such as Elapidae, Viperidae, and Crotalidae (Cohen and Levi-Montalcini, 1956; Lipps, 2002). It is therefore of interest to investigate the presence or absence of NGF in sea snake venoms. In order to investigate the presence or absence of NGF, five sea snake venoms were selected. Lapemis hardwickii (Hardwick's sea snake) and Acalyptophis peronii venom were obtained from the Gulf of Thailand. Hydrophis cyanocinctus (common sea snake) and Enhydrina schistosa (beaked sea snake) venom were obtained from the Strait of Malacca. Laticauda semifasciata (broad band blue sea snake) venom was also examined and the venom was obtained from Gato Island in the Philippines.

The neurotoxic effects of the Synanceia verrucosa venom were investigated in rodents. After intracranial injection in mice (50-125 ng/g), venom induced constant symptoms such as ataxia, circling, partial or complete reversible limbs paralysis, scratching, rolling, sleep-like periods and violent clonic seizure conducing in few seconds to death. EEG alterations occurring in rat brain after intracerebroventricular injection (50-100 microg) were precised. An initial phase was characterized by short repetitive tonic seizure periods together with a significant rise of the relative power in the delta band, no significant modification of the theta II rhythm (4-7 Hz), a decreasing of energy in theta I (7-12 Hz) and 15-40 Hz bands. A second phase was characterized by a marked generalized slowing with transient drastic decreasing of the amplitude and flattening of cortical EEG (comatose state) as the main elements. Propanolol did not reverse the EEG effects of the venom except a slight decrease of the slow wave amplitude. Previous intracerebroventricular administration of a K+(ATP) blocker generally decrease the delay of death. Histopathologic examination of the brain of surviving animals did not reveal any microscopic lesions. These results suggest (1) a complex mechanism of the venom in its neuropathologic expression; (2) at the doses tested, symptoms are not related to adrenergic pathways, K+(ATP) channel opener (verrucotoxin) is not implied in the neurotoxic effect, and the effect of the venom, which not affecting the theta II rhythm, seemed not to be exerted through cholinergic pathway.

Immunization with Bungarus candidus venom was performed in four rabbits at high dose (initial dose, 75 microg/kg) and low dose (initial dose, 50 microg/kg). Each dose group consisted of two rabbits; one rabbit received the venom subcutaneously (s.c.) and the other intradermally (i.d.). The venom was injected as emulsified solutions with the same volume of Freund's complete adjuvant until the 4th immunization, thereafter as plain solutions. By stepwise increments of the immunizing dose, the higher dose group received a dose of 200 microg/kg and the lower dose group 150 microg/kg after the 5th immunization, respectively. Thereafter, seven additional immunizations were performed within six months. All rabbits were sacrificed two weeks after the last immunization (12th). Antilethal activity of the immunized antisera thus obtained was determined not only with the homologous venom but also with two heterologous venoms from Bungarus fasciatus and Bungarus flaviceps. Immunodiffusion analysis was also performed with these venoms. The results obtained in this pilot trial provided useful information for production of Malayan krait antivenom at Queen Saovabha Memorial Institute.

A soluble form of alkaline phosphatase (ALP) has been identified and purified from Walterinnesia aegyptia venom using an HPLC system Gold 126/1667 equipped with Protein PAK 125 and Protein PAK 60 columns. The enzyme was purified 3.4 fold over crude venom with a yield of 37.3%. On SDS-PAGE under non-reduced conditions the purified enzyme showed three bands of 212 kD, 80 kD, and 55 kD. However, under reducing conditions, the enzyme showed two bands of 80 kD and 55 kD. The specific activity of ALP was 24 U/mg with p-nitrophenylephosphate as the substrate. During isoelectric focusing experiments the ALP exhibited two bands focused at pH 6.2 and 6.8, which suggests that either the enzyme exists as two different isoforms or the two bands in IEF may be two subunits of 80 kD and 55 kD. The kinetic parameters (Km and Vmax) and IC50 of ALP inhibition by L-phenylalanine, L-leucine, imidazole, caffeine, orthophosphate and permanganate were also investigated in the present study. Zinc and cyanide ions at a concentration of 15 mM and 10 mM, respectively, completely inhibited the activity of W. aegyptia ALP.

Toxins are non-replicating agents of biological origin. They are non-infectious, non-contagious, and non-curable by antibiotics or chemotherapeutic agents. However, individuals can be protected by vaccination. The multifactorial nature of virulence of toxin and toxin producers, produces comparative and cooperative pathogenesis, and this makes studies all the more difficult. Antibody raised against all components helps in this pursuit. The toxins have been classified into seven different classes and over 44 bacterial toxins have been discussed. The botulinum toxin is by far the most toxic substance in the world. All the toxins produced are either secreted out, called exotoxins (proteins), or are entrapped in the cell membrane, called endotoxins (lipopolysaccharides). These toxins are di-chain molecules, internalized into the cell by receptor mediated endocytosis, and ADP-ribosylation is the most common mode of action. The toxins produced by bacteria are enterotoxins, neurotoxins, cytotoxins, lysins (e.g., hemolysin), gangrene producing toxins. However, a single bacteria often produces more than one toxin. Bacterial toxins, which are primarily harmful, are also being used for the cure of cancer, killing of mosquito larvae, understanding of basic sciences like ADP-ribosylation, etc.

The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons. There is little information on the cellular processing of proteins delivered by linkage to TTC. We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E. coli. Binding, internalization, and cleavage of this protein during prolonged incubation with fetal cortical neurons or cells of the N18-RE-105 line was evaluated by immunoblot analysis, ELISA, and immunocytochemistry. Hybrid proteins were bound and internalized in a manner very similar to TTC. Internalized proteins showed long-term stability within cells, and were degraded into predictable large protein fragments in both cell types. Fragments that were cleaved away from the TTC domain were released into extracellular fluid after internalization. Proteins coupled to TTC share its long-term stability after cellular internalization. After internalization, dissociation of proteins linked to TTC facilitates their release from the cell, but not into other cellular compartments such as the cytosol. TTC linked proteins are probably enclosed within a stable endosomal compartment throughout their cellular lifetime.

An epidemic investigation was carried out on freshwater puffer poisoning incidents in Bangladesh from April 1988 to May 1996. A lot of information on 10 poisoning cases involving 55 victims was collected through newspapers, interviewing the victims and their families, concerned hospital sources or questionnaires to them. Symptoms of the victims were partly similar to those caused by paralytic shellfish poison (PSP) or tetrodotoxin (TTX). Among them, however, muscle pain, discharge of black urine, and longer recovery time are clearly different. Further, serum creatine phosphokinase (CPK) values were found to be higher (230-450 and 298-430 IU/l) than normal values in two cases. From these different symptoms and high CPK values, it can be predicted/assumed that present freshwater puffer toxin is implicated in not only PSP, but also other toxin(s).