An L-amino acid oxidase (LAO), designated as TJ-LAO, was purified to homogeneity from the venom of Trimeresurus jerdonii by Sephadex G-100 and Q Sepharose HP chromatography. The molecular weight of this enzyme was 110 kD as estimated by analytical gel filtration and was 55 kD by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two subunits. The enzyme has an absorption spectrum characteristic of flavoproteins, containing 2 moles of FMN per mole of enzyme. The N-terminal sequence of TJ-LAO shares high homology with other viperid snake venom LAOs. Homology with elapid venom LAO is lower. TJ-LAO inhibited the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus megaterium. The antibacterial effect associated with LAO activity was elminated with the addition of catalase. Platelets in platelet-rich plasma aggregated upon the addition of TJ-LAO. The enzyme-induced aggregation was inhibited by catalase, suggesting formation of H2O2 was essential for TJ-LAO to induce platelet aggregation. These results showed H2O2 formation is important for the biological effects of LAO.
The effects of sea snake venom (SSV) on renal function were studied in two groups of anesthetized experimental dogs pretreated with intravenous infusion of 4.2 gm% NaHCO3 solution. Animals were envenomated by intramuscular injection of SSV at a dosage of 0.34 mg/kg. Systemic hemodynamics showed no significant changes except for a tendency of decrease in cardiac output (CO). The glomerular filtration rate (GFR), the rate of urine flow (V) and effective renal plasma flow (ERPF), and effective renal blood flow (ERBF) significantly decreased, while filtration fraction (FF) significantly increased at 180 min after envenomation. Envenomated animals showed a reduction in renal fraction (RF), while renal vascular resistance (RVR) increased stepwise throughout the experimental periods. Animals pretreated with sodium bicarbonate showed no significant changes of CO, TPR MAP, HR, and packed cell volume (PCV) while receiving sea snake venom. Animals pretreated with sodium bicarbonate showed no changes in GFR, ERPF, ERBF, RF, and RVR after envenomation. The rate of urine flow markedly increased in envenomated animals which received pretreatment with bicarbonate. After envenomation alone, there were no differences in the plasma concentration of sodium (PNa) and chloride (PCl) as compared to the control value, whereas the plasma concentration of potassium (PK) increased at 180 min after envenomation. Animals pre-treated with bicarbonate showed a stepwise increase in both UNaV, FE(NA), U(Cl)V, and FE(Cl) accompanying SSV injection. Neither PNa nor PCl were affected, while PK significantly decreased in animals given SSV with bicarbonate loading. UKV and FEK increased stepwise in envenomated animals treated with bicarbonate throughout the period of study. All groups of animals given SSV, with or without NaHCO3 infusion, showed a marked elevation of the concentration of urinary myoglobin (U(Mb)), plasma lactate dehydrogenase (LDH), and plasma creatine phosphokinase (CPK) throughout experimental periods. The urinary myoglobin excretion markedly increased in animals after SSV injection accompanied by NaHCO3 infusion. It can be concluded that large amounts of myoglobin present in the renal tubules in envenomated animals can precipitate, particularly under acidic conditions, resulting in increased intratubular pressure and subsequently decreased renal hemodynamics including GFR and ERBF. An infusion of NaHCO3 to render urine more alkaline could have a protective role against depression of renal function following sea snake venom administration.
A thrombin-like enzyme with a new amino-terminal sequence was isolated from the venom of Trimeresurus flavoviridis using Q-Sepharose, CM-Cellulose, and HW55 column chromatographies. Homogeneity was confirmed by the formation of a single band in polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 29,000 Da. This thrombin-like enzyme was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), and dithiothreitol (DTT) suggesting that serines and disulfide bonds are involved in the expression of the enzyme's clotting activity. This thrombin-like enzyme hydrolyzes the Aalpha-chain and Bbeta-chain of bovine fibrinogen. The enzyme was stable to heat treatment.
Forty-two and twelve specimens of puffers Tetraodon ocellatus and T. nigroviridis were, respectively, collected from the aquaria in Taiwan and determined for toxicity by using tetrodotoxin bioassay. It was found that T. ocellatus contained moderate amounts of toxin in skin and viscera, and the highest toxicity scores were 432 MU/g skin and 212 MU/g viscera. The specimens of T. nigroviridus contained weak amounts of toxin in skin, and the highest toxicity score was 124 MU/g. The toxin was partially purified from the toxic specimens of each species by ultrafiltration using a YM-1 membrane, followed by chromatography on Bio-Gel P-2 column. Analyses by electrophoresis, thin layer chromatography, and high performance liquid chromatography showed that the toxin from both species was composed of tetrodotoxin and anhydrotetrodotoxin.
Quantitative determination of newly reported enzymes activity in the crude skin toxin (CST) of catfish revealed highest activities of hyaluronidase and lipase, lesser activities of phospholipase A2, lactate dehydrogenase (LDH), cholinesterase (CE), alkaline phosphatase (ALP), and aspartate transaminase (AST), and least activities of proteinase and 5-nucleotidase (5'-NT). The CST has a hemolytic activity of 54% and no ichthyotoxicity up to 500 ug/ml. The chosen dose of CST (LD12.5) showed a potential cytotoxic activity against solid Ehrlich carcinoma-bearing mice demonstrated by an increase in the mean survival time (238.8%) and tumor growth inhibition ratio (T/C) of 73%. The CST ameliorated the relative weights of heart and liver after three weeks, while modulating the elevation in the relative spleen weight throughout the treatment periods (three, six, and nine weeks). The levels of serum triglyceride, total cholesterol, and liver total lipids were normalized after three weeks, whereas the serum albumin and hepatic glycogen concentrations, as well as ALT, AST, 5'-NT, and G-6-Pase activities were ameliorated after 6 weeks. Serum levels of glucose, LDH, and creatine kinase (CK) activities were significantly modulated throughout the treatment periods. Histological examinations of the tumor and liver tissues of treated tumor-bearing animals were carried out. Tumor tissues showed many cytolytic and cytopathic changes after treatment, while liver tissues showed moderate dysplastic changes after six weeks of treatment, which became more marked after nine weeks.
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.
This study was performed to assess the ability of polyvalent snake venom anti-serum, produced by the Egyptian Organization for Biological Products & Vaccines (VACSERA), to neutralize several toxic activities of snake venoms, not only of those included in the antivenom mixture, but also some additional venoms of snakes from Egyptian, African, and Middle Eastern habitats. In general, the results revealed that polyvalent snake venom anti-serum from VACSERA is highly effective in neutralizing Egyptian snake venoms, especially Naja haje, Naja nigricolles, Naja pallida, Cerastes cerastes, Cerastes cerastes cerastes, Cerastes vipera, Pseudocerastes persicus fieldi, and Walterinnisia egyptia. The antivenom was also effective against Naja haje, Walterinnisia egyptia, and Bites aritans from Saudi Arabia. High activity was obtained against venoms from Naja haje, Naja nigricolles, and Naja pallida of Sudan, as well as the African Naja melanoleuca, Naja mossambica, Naja naja oxiana, Bites gabonica, and Vipera lebetina. Only moderate effectiveness was obtained with Echis coloratus and Echis carinatus, and the polyvalent antiserum was ineffective against the venom of Naja nivea.
Water-soluble skin secretions of salamander Tylototriton verrucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. verrucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A2 activity; however, no acetylcholine esterase activity was detectable under the assay conditions.
Several biochemical and biological activities such as phospholipase A2, arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA2) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA2 had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA2 was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.
Three short-chain neurotoxins named NT-1, NT-II, and NT-III were purified from the venom of Naja kaouthia, a snake distributed throughout the south of Yunnan province, China, by a series of chromatographic steps, including an FPLC Resource S column. Their molecular weights, determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, were 6952.19 Da, 6854.92 Da, and 6828.80 Da, respectively. NT-I consisted of 62 amino acid residues, and the other two consisted of 61 amino acid residues, including 8 cysteines. After hydrolysis by endoproteinase Glu-C, their primary sequences were determined. A test of their activities demonstrated that they effectively inhibited muscle contractions induced by electric stimulation. Furthermore, the extent of inhibition caused by NT-II and NT-III was less than that of NT-I. The IC50s were 0.04 microg/ml, 0.20 microg/ml, and 0.23 microg/ml for NT-I, NT-II, and NT-III, respectively. Compared with NT-II and NT-III, the higher activity of NT-I may be a result of the amino acid residue substitution Ile36 to Arg36.
A three-year surveillance program assessed the extent of mycotoxin contamination of key foods and feeds grown in Bangladesh. The study also included groundnuts utilized as snack food. In the first two phases of the program the samples collected were analyzed only for aflatoxins, but in the third phase, as well as for aflatoxins, samples were tested for the presence of fumonisin B1, ochratoxin A, zearalenone, deoxynivalenol, and T-2 toxin. Of the foods and feeds tested, the incidence of aflatoxin contamination varied from low (rice collected from farmers' stores, 8%) to high (maize, 67%). However, both the average total aflatoxin contents (< 1.0 microg/kg) and the maximum aflatoxin B1 contents (< or = 5.0 microg/kg) recorded for pulses, rice and its various products, and wheat were low. On the other hand, the levels of contamination of maize, roasted and raw groundnuts, and poultry feed were considerably higher, with average total aflatoxin B1 contents of 33, 13, 65, and 7 microg/kg, respectively, and maximum aflatoxin B1 contents of 245, 79, 480, and 160 microg/kg, respectively. Fumonisin B1, ochratoxin A, zearalenone, deoxynivalenol, and T-2 toxin were found, to any significant extent, only in some of the maize samples tested, always accompanied by aflatoxins. One sample of maize contained five mycotoxins, namely, the aflatoxins, fumonisin B1, deoxynivalenol, zearalenone, and ochratoxin A. In a limited trial using hospital staff in Dhaka, the analysis of the aflatoxin-albumin adduct in serum showed that approximately half of the test group had been recently exposed to low levels of aflatoxins.
The main objective of the present work was to obtain a stable and highly purified scorpion antitixon, rich in specific antibodies, free of immunologically irrelevant plasma proteins or gross proteins.Our results indicate that the most potent yielded and purified F(ab')(2) antivenom preparation was obtained when the first discarded precipitate was washed with 14% ammonium sulphate saline; then after the second addition of ammonium sulphate, the mixture was stirring overnight followed by precipitation of most non-immunoglobulin proteins with the aid of caprylic acid to produce antivenom rich in specific antibodies with higher yield and potency compared to the method commonly used.
The capacity of Thai green pit viper antivenom raised to Trimeresurus albolabris to neutralize the venoms from six species of Trimeresurus sp. in Thailand has been examined. They were Trimeresurus albolabris, T. macrops, T. popeiorum, T. hageni, T. purpureomaculatus, and T. kanburiensis. The antivenom neutralized lethal and hemorrhagic activities of all these venoms. The capacity of antivenom to neutralize lethal toxicity of the venom was expressed as the amounts (mg) of snake venom neutralized by 1 ml of the antivenom. The largest capacity was found with the homologous venom. Results of immunodiffusion, immunoblotting, and antigen-antibody complex formation experiments supported the results of neutralization experiments. Several biological activities of the Trimeresurus venoms were also examined and compared. They were lethal, hemorrhagic, proteolytic, phospholipase A, arginine ester hydrolyse, and thrombin activities. There was no correlation between the ratios of lethal toxicity and hemorrhagic activity, lethal toxicity and phospholipase A activity, as well as hemorrhagic activity and proteolytic activity.
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.
The presence of nerve growth factor (NGF) is reported in salivary glands of mice and sex organs of various male animals. NGF level is reported to be higher in salivary glands of male mice in comparison to female. This investigation reports the presence of NGF in major organs of Balb/c mice showing quantitative differences in NGF according to sex and age. Organs of Balb/c mice from ages 2 weeks to 9 months were tested for NGF content by immunological tests enzyme-linked immunosorbent assay (ELISA). Male and female mice for each age group were from the same litter. It was revealed that NGF levels increased with age, reaching the peak at 4 weeks for male mice versus 3 months for female for similar organs. There were consistently lower levels of NGF in the organs of female mice in comparison to their male siblings. Most decreased levels of NGF were observed in organs of mice with respect to heart, salivary glands, and sex organs, ovaries versus testes. The presence of NGF was further revealed in non-neural organs. NGF based maturity was reached at 4 months for male mice versus 6 months for females.
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.
Among several batches of clams harvested in Tunisia and imported to France, a small number of them were found to be neurotoxic to mice (intraperitoneal injection) as determined by the diarrhetic shellfish poisoning (DSP) bioassay developed by Yasumoto et al. (1978). The present study was conducted to confirm the nature of the toxic agent, suspected to be gymnodimine. Liquid chromatography tandem mass spectrometry analyses unequivocally revealed the presence of gymnodimine in the shellfish, making Tunisia the second country, after New Zealand, where shellfish contamination with this phycotoxin is reported. Gymnodimine B and C analogues were not detected in the clam samples. Gymnodimine preferentially accumulates in the digestive gland of the Tunisian clams, although substantial amounts are also found in the meat.
Attempts were made to elucidate the different responses of shellfish to paralytic shellfish poison (PSP) and the PSP donor Alexandrium minutum T1. Five species of edible bivalves (Crassostrea gigas, Meretrix lusoria, Mytilus edulis, Ruditapes philippinarum, and Soletellina diphos) were collected and examined for susceptibility to PSP and PSP donor. It was determined that all five bivalves had low susceptibility to PSP following an intramuscular injection (> 300 MU/20 g). The abnormal effects on bivalves were species-specific and varied with the concentration of A. minutum T1. Judging from the LC50 data (medium lethal concentration), the resistance of bivalves to the toxic dinoflagellate was as follows (least to most resistant): C. gigas < R. philippinarum < M. lusoria < M. edulis, S. diphos. With the exception of S. diphos, the bivalves accumulated very little toxin (< 2 MU/g edible tissue) when they were exposed to 10(7) cells/L of A. minutum for four days. The toxin levels in S. diphos increased with exposure time to the toxic dinoflagellates and accumulated primarily in the digestive gland (88-100%), followed by the gill (0-10%), and other organs (0-8%). Although the concentrations of toxin components in the digestive gland were found to be variable during the exposure period, the toxin profile in the digestive gland of S. diphos during the early exposure period was similar to that of A. minutum. Moreover, toxin components in the gills and in other organs were retained at near constant concentrations during the exposure period.
Heptyl prodigiosin was purified from a culture of alpha-proteobacteria isolated from a marine tunicate collected in Zamboanga, Philippines, as part of a program to screen natural products for antiparasitic activity. An in vitro antimalarial activity similar to that of quinine was found against the chloroquine-sensitive strain Plasmodium falciparum 3D7. The in vitro antimalarial activity was about 20 times the in vitro cytotoxic activity against L5178Y mouse lymphocytes. A single subcutaneous administration of 5 and 20 mg/kg significantly extended survival of P. berghei ANKA strain-infected mice but also caused sclerotic lesions at the site of injection. A single administration by gavage of 50 mg/kg did not increase survival time. The compound was not found to be mutagenic using in vitro micromethods for the Ames Salmonella typhimurium assay and the micronucleus assay using L5178Y mouse lymphoma cells.
