Journal of natural toxins最新文献

Biological activities in Vipera a. aspis and V. a. zinnikeri venom were investigated and compared. Phospholipase A2 and lethal activities were found to be much higher in V. a. zinnikeri venom; the LD50 values for V. a. aspis and V. a. zinnikeri crude venom were 0.55 and 0.35 microgram/g, while PLA2 activities were 25.4 and 41.8 unit/mg, respectively. Other enzymatic and pharmacological activities investigated were similar in both venoms, suggesting that PLA2s might be responsible for the higher toxicity of V. a. zinnikeri venom. PLA2s contained in both venoms were compared immunologically, and the highly lethal phospholipase A2 (PLA2-I) purified from V. a. zinnikeri venom was not found in V. a. aspis venom as determined by antiserum for purified PLA2-I. The toxic effect of V. a. zinnikeri venom was inhibited by anti-PLA2-I, while the same antiserum could not prevent lethality by V. a. aspis venom. These results indicate that PLA2-I in V. a. zinnikeri venom possesses an important role in the lethal activity of this venom. Although V. a. zinnikeri is found in a defined area of France, it is possible that its lethal venom component developed differently than that of V. a. aspis.

Mussel samples from four locations along the Norweigian coast were extracted by methods for diarrheic shellfish toxins (DST) and tested by chemical and biological methods, including histopathology. All samples had previously been found to be highly toxic in mice, with symptoms indicating the presence of non-diarrheagenic toxins in the mouse bioassay. Chemical analysis revealed that the DST okadaic acid (OA) and dinophysistoxin-1 (DTX1) were present each one in one sample, but only a minor part of the total toxicity could be attributed to these toxions. In the other two samples, OA and DTX1 were absent. Incubation of the mussel extracts from all four samples with freshly prepared hepatocytes indicated the presence of unknown toxin(s) which may not be classified within the DST complex. Purified mussel samples were given to baby mice both via intraperitoneal (i.p.) injections and by oral intubation. Oral toxicity was about 25-50 times lower than toxicity obtained by i.p. injections, a result in accordance with acute toxic properties of many toxins. Risk assessment of the unknown toxin(s) requires chemical identification, but the preliminary results obtained indicate a large margin of safety, based on the large amounts of mussel extracts necessary to yield toxic effects in the intestine and liver in experimental animals upon oral exposure versus human intake.

Cleft palate and minor front limb contractures were induced in calves by maternal ingestion of the piperidine alkaloid-containing lupines, Lupinus formosus and L. arbustus. Crooked calf disease, which includes an occasional cleft palate, is a congenital condition of widespread occurrence in cattle in the western U.S. and Canada. It is known to occur after maternal ingestion of certain species of Lupinus during specific gestational periods. Although many lupine species contain quinolizidine alkaloids including the teratogenic alkaloid anagyrine, L. formosus and L. arbustus produce piperidine alkaloids including the reported teratogen ammodendrine. In addition to ammodendrine, L. formosus contains both N-acetyl hystrine and N-methyl ammodendrine, whereas L. arbustus contains ammodendrine, trace amounts of N-methyl ammodendrine, and no N-acetyl hystrine. L. formosus and L. arbustus were fed to pregnant cows at equivalent ammodendrine doses during a 10-day period from days 40-50 of gestation. One calf from a cow fed L. formosus had a full cleft palate. Embryonic death and resorption of one fetus and minor front limb contractures (arthrogryposis) in another calf occurred with two cows fed L. arbustus. Alkaloid analysis of blood samples taken during the feeding period, and up to and including 48 hours after the last dose, demonstrated comparative plasma elimination times with N-methyl ammodendrine > ammodendrine > N-acetyl hystrine. The objectives of this experiment were to: 1) determine if N-acetyl hystrine is a potential teratogen; and 2) define the narrow cleft palate induction period in cows.

The efficacy of the Thai green pit viper antivenom to neutralize lethal, hemorrhagic, and enzyme activities of Trimeresurus venoms was examined using venoms of Trimeresurus albolabris, T. macrops, and T. flavoviridis (Japanese Habu). Antivenom against Japanese Habu venom was also used to study immunological cross reactivity among Trimeresurus venoms. Thai green pit viper antivenom was comparably effective to Habu antivenom to neutralize all activities. Distinct cross neutralization was demonstrated indicating the presence of genus specific protection by Thai and Japanese Trimeresurus antivenoms. Results of immunoblotting analyses indicated that Thai and Japanese Trimeresurus venoms contain many cross-reactive protein components.

A nonglycoprotein-like fibrinolytic enzyme ((FIB-I) was purified from the crude venom of Agkistrodon acutus by CM-Sepharose CL-6B and DEAE-Sepharose CL-6B ion exchange chromatography and then by FPLC through Superose 12 gel filtration. Its molecular weight is about 23 kDa and isoelectric point is near 6.0. It not only has fibrinolytic and caseinolytic activity, but also can hydrolyze BAEE. The local hemorrhagic activity was found in mice after the subcutaneous injection of this enzyme. EDTA can inhibit its fibrinolytic activity completely, but PMSF and arrowhead proteinase inhibitor have no such obvious inhibitory effect, thus implying that FIB-I is a metalloproteinase. The N-terminal ten amino acid residues 'STEFQRYMEI' of FIB-I was elucidated. A full-length cDNA gene of this enzyme was cloned by using RT-PCR from the total RNA extracted from the snake venom gland and FIB-I was expressed in E. coli. Having analyzed the sequence, we found that it had a typical zinc-chelating characteristic as 'HEXXHXXGXXHD.'

The biosensor consisted of a sodium electrode and covered with the frog bladder membrane within a flow cell was tested for the estimation of tetrodotoxin (TTX) and saxitoxin (STX). This sensor was applied to detect very low amounts of the Na+ channel blockers, STX and TTX, in different shellfishes and swellfishes. A good agreement was obtained between TTX activities determined by mouse assay and amounts of Na+ channel blockers estimated by frog membrane sensor. The lowest level of TTX (fg) that can be determined by frog membrane sensor does not cause human poisoning. The channel blockers in short-necked clam, which was assumed to be STX, were monitored by this sensor continuously every week for one year. It was discovered that the STX content increased from July until September and then decreased from October until March. The biosensor proposed here may be used for the estimation of STX and TTX conventionally in the future.

Homogeneous preparation of nerve growth factor (NGF) was isolated in purity by two steps HPLC fractionation from venoms of snakes belonging to the major families: Crotalidae, Elapidae, and Viperidae. Biological activity of NGF was tested on PC12 cells for neurite outgrowth and molecular weights were determined by PAGE. Antisera raised against NGFs in Balb/C mice. Immunological cross reactivity for antisera was assayed by ELISA and immunoprecipitin tests. HPLC profiles for the venoms from the species belonging to the same family were identical. The biological and immunological properties of NGFs from different species of snake belonging to the same family were also found to be identical. However, NGFs of venoms from different families of snakes showed differences in properties. Neurite outgrowth on PC12 cells due to NGF from the family Elapidae, especially the cobra species, was greater than NGF from the venoms of Crotalidae and Viperidae, with the exception of N. n. nivea which showed poor activity and C. polystictus of Crotalidae family having very good activity.

A simple procedure to prepare the toxic components from Naja kaouthia venom for use as immunogens has been studied. The aim was to produce serum rich in antitoxins. By heating the venom (1-6 mg/ml) at 100 degrees C for 10 min at pH 5.0, at least 10 proteins with MW greater than 25,000 daltons were precipitated and removed. The toxic components, i.e., postsynaptic toxins, Direct Lytic Factor (DLF), and phospholipase A2 were relatively stable to this treatment; however, their activities were progressively lost as the heating time was prolonged. The LD50S of the heated (100 degrees C, 10 min) and the unheated venom were 0.37 and 0.325 mg/kg, respectively. As compared to the unheated venom, immunization of rabbits with the heated venom resulted in a 3.38-fold increase in precipitable antibodies against N. kaouthia toxin 3 and a 1.85-fold increase in neutralizing capacity. This toxin preparation should be useful as an immunogen or as a starting material for chemical modification prior to immunization in the production of potent therapeutic antiserum.

The effects of Russell's viper venom (RVV) on human erythrocytes were studied in vitro with respect to packed cell volume (hematocrit), the erythrocyte morphology, and the effect of antivenom. Venom at various dosages ranging from 50 ng to 120 micrograms increased hematocrit significantly. The maximal effect was detected at 800 ng of venom. The biconcave erythrocytes shown by scanning electron microscopy became sphero-echinocytes. Such altered morphology was observed immediately at 1 minute and reached maximum at 30 minutes. The mild degree of erythrocyte deformation was observed at 60 and 120 min with 100 ng of RVV. There were no morphologic changes when ethylenediaminetetracetate (EDTA) was used as an anticoagulant or when plasma was substituted by isotonic saline, acetar, albumin added acetar solution. Phospholipase A2 at equivalent dose as compared to the venom could also produce the sphero-echinocytosis. The phospholipase A2 inhibitor, p-bromophenacyl bromide markedly reduced the degree of RVV induced sphero-echinocytosis. Verapamil, a phenylalkylamine calcium channel blocker, could not prevent the RVV induced sphero-echinocytosis. Although antivenom could not reverse the RVV induced sphero-echinocytosis, it minimized these effects. The RVV induced sphero-echinocytosis is likely caused by phospholipase A2. Calcium and some plasma factors are required for this process. Early treatment with antivenom plays some role in prevention of RVV induced sphero-echinocytosis which may reduce hypoxic cell injury.

A kinin-releasing enzyme was isolated and characterized from the venom of Trimeresurus okinavensis (himehabu) using Sephadex G-100, DEAE-Cellulose, and CM-Cellulose column chromatographies. The kinin-releasing enzyme was homogeneous as demonstrated by a single band on polyacrylamide gel electrophoresis, and isoelectric focusing. The enzyme possesses a molecular weight of 31,000 Da and isoelectric point of 8.2 and consists of 312 total amino acid residues. Specific esterolytic activities of the kinin-releasing enzyme on N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethylester (BAEE) were determined to be 235.3 and 111.3 mumol/min/mg, respectively. The enzyme was inhibited by p-APMSF (p-amidinophenylmethanesulfonyl fluoride hydrochloride) and benzamidine. Additionally, the enzyme was found stable to heat treatment. The enzyme cleaved a kininogen analog with the release of bradykinin, resulting in an immediate drop in blood pressure, and contractions of the rat uterus were also observed.