Journal of natural toxins最新文献

Research on teratogenic plants started at the USDA-Agricultural Research Service-Poisonous Plant Research Laboratory in the mid 1950s when Dr. Wayne Binns, Director of the laboratory, was asked to investigate the cause of a cyclopian facial/skeletal birth defect in lambs. Dr. Lynn F. James joined the staff shortly after. These two people worked as a team wherein most planning was done jointly with Binns supervising most of the laboratory work and James the field studies. It was determined that when pregnant ewes grazed Veratrum californicum on day 14 of gestation a significant number of lambs had the cyclopic defect. Skeletal and cleft palate birth defects in calves was associated with pregnant cows grazing certain lupine species during 40-70 days of gestation. Shortly thereafter research work was initiated on locoweed which caused abortions, wasting, right heart failure, skeletal birth defects, and fetal right heart failure. Dr. Richard F. Keeler, a chemist who joined the staff in the early 1960s, isolated and characterized the teratogens in V. californicum as the steroidal alkaloids cyclopamine, jervine, and cycloposine. He also described the teratogen in lupines as the quinolizidine alkaloid anagyrine and the piperidine alkaloid ammodendrine. Drs. Russell Molyneux and James identified the toxin in locoweed as the indolizidine alkaloid swainsonine. In 1974 the editor of Nutrition Today (Vols. 9 and 4) wrote "The idea that birth defects occurring in humans may be in some way related to diet is not widely held ..." Dr. Lynn James pointed out in this issue that such defects in animals can be produced with absolute predictability and regularity by foods ordinarily beneficial to livestock. Management strategies have been developed to prevent or minimize the economic impact of the cyclopian lamb and the crooked calf condition on livestock producers and well on the way to doing the same with locoweed. It is of interest to note that livestock research on Veratrum, lupines and locoweed and toxins therefrom are now significant research tools for specific human health problems.

More than 350 PAs have been identified in over 6,000 plants in the Boraginaceae, Compositae, and Leguminosae families (Table 1). About half of the identified PAs are toxic and several have been shown to be carcinogenic in rodents. PA-containing plants have worldwide distribution, and they probably are the most common poisonous plants affecting livestock, wildlife, and humans. In many locations, PA-containing plants are introduced species that are considered invasive, noxious weeds. Both native and introduced PA-containing plants often infest open ranges and fields, replacing nutritious plants. Many are not palatable and livestock avoid eating them if other forages are available. However, as they invade fields or crops, plant parts or seeds can contaminate prepared feeds and grains which are then readily eaten by many animals. Human poisonings most often are a result of food contamination or when PA-containing plants areused for medicinal purposes. This is a review of current information on the diagnosis, pathogenesis, and molecular mechanisms of PA toxicity. Additional discussion includes current and future research objectives with an emphasis on the development of better diagnostics, pyrrole kinetics, and the effects of low dose PA exposure.

Thai commercial antivenom raised to Bungarus fasciatus venom neutralized the lethal activity of all Thai Bungarus venoms tested in in vitro neutralization experiments. The neutralizing capacities against B. fasciatus and B. candidus venoms were almost the same, but that against B. flaviceps venom was significantly greater. The efficacy of the antivenom was confirmed in in vivo neutralization experiments also. Results of immunochemical analyses supported results of the animal experiments suggesting the presence of genus specific neutralization.

Type A Clostridium botulinum, the causative agent of the food poisoning botulism disease, secretes botulinum neurotoxins along with seven neurotoxin associated proteins (NAPs). The function of NAPs has been shown to protect the neurotoxin from acidity, heat, and proteolytic attack in the environmental and gastrointestinal tract during the toxicogenesis of the botulism disease. One of the NAPs, purified from type A botulinum neurotoxin complex, showed hemagglutination activity. A direct interaction has been demonstrated between purified NAP, a 33-kDa hemagglutinin or Hn-33, and the neurotoxin by using Sephadex G-200 column chromatography. Furthermore, Hn-33 has complete resistance against proteolytic attack at pH 2.0 as well as at normal physiological pH. We have investigated digestion of the neurotoxin in the presence and absence of Hn-33. The neurotoxin alone has been found to be more susceptible to the enzymatic digestion than neurotoxin with Hn-33. The presence of Hn-33 changes the proteolytic fragmentation pattern of the neurotoxin. It seems that Hn-33 protects the neurotoxin from proteolysis either by structural modification of the neurotoxin or by blocking the protease accessible sites of the neurotoxin.

Clostridium botulinum neurotoxin acts on nerve endings to block acetylcholine release. Binding of the neurotoxin to a membrane receptor through its heavy chain is the first essential step in its mode of toxin action. Type E botulinum neurotoxin (BoNT/E) or type A botulinum neurotoxin (BoNT/A) receptor was purified from rat brain synaptosomes employing a neurotoxin affinity column chromatography. The protein fraction eluted from the affinity column with 0.5 M NaCl contained a 57 kDa protein as a major eluant. Immunoblotting the eluant with anti-synaptotagmin antibodies revealed that the 57 kDa protein was synaptotagmin I. Rat synaptotagmin I has been suggested as the receptor for BoNT/B (Nishiki et al., J. Biol. Chem. 269, 10498-10503, 1994) in rat brain. In this study, binding of BoNT/A and BoNT/E to synaptotagmin I was studied by a microtiter plate-based method. Binding of synaptotagmin I to BoNT/A coated on the plate was competitively reduced upon preincubation of the proteins with BoNT/E, suggesting a competitive binding of BoNT/A and BoNT/E to the receptor. Taken together, these results suggest that the same receptor protein binds to all three BoNT serotypes tested.

CRM45 is a mutant form of diphtheria toxin (DTx) that lacks a 17-kDa carboxyl-terminal segment of the receptor-binding B subunit (DTB). The missing segment is a discrete structural domain of DTB that normally rests against the NAD binding pocket of the enzymically-active A subunit (DTA). Proteolytic cleavage and disulfide bridge reduction in the DTA-DTB linker region of DTx are required for optimal ADP-ribosylation of elongation factor 2 (EF-2). Here, we show that cleaved and uncleaved preparations of X-ray crystal grade CRM45 both exhibit an ADP-ribosyltransferase activity similar to that of cleaved DTx. Crystal-grade preparations of CRM45 also display a potent deoxyribonuclease activity. However, as observed with DTx, cleavage and reduction of CRM45 are not required for expression of this nuclease activity. After SDS-PAGE in a gel that contains DNA embedded in the matrix, renaturable Ca++/Mg(++)-dependent nuclease-active bands co-migrate with intact CRM45 (45 kDa) as well as with the DTA subunit (24 kDa) of CRM45. Because the 45-kDa nuclease-active band is unique to the CRM45 form of DTx, it offers direct proof that this activity is intrinsic to the DTA domain of DTx and its homologues.

Persistent intramammary infections of dairy cows with Staphylococcus aureus may involve immunosuppression mediated by bacterial toxins such as enterotoxins and other super-antigens (SAgs). Previously we found that stimulation of bovine PBMC with staphylococcal enterotoxin C (SEC) induced a unique phenotype of activated CD8+ T cells expressing a newly identified activation molecule, ACT3. In the present study we found that SEC induced the expression of interleukin (IL)-4 and IL-10 mRNAs, two cytokines associated with type 2 responses. Elevated levels of IL-4 and IL-10, observed between day 0 and day 4 of culture, were associated with temporary inhibition of proliferative responses of T cells, evidenced by a decrease in numbers of CD4+ T cells and a small increase in numbers of CD8+ T cells. Vigorous proliferation of T cells occurred between days 4 and 7 of culture and with a bias towards CD8+ T cells. Acquisition of the ACT3+ phenotype by CD8+ T cells was preceded by induction of IL-4 mRNA. Thus, in the bovine system, SAgs may hinder protective responses by inducing type 2 cytokines, which interfere with immune clearance of many microbial pathogens. The results of the study are consistent with the hypothesis that SAgs are involved in immunosuppression, and suggest possible immunomodulatory mechanisms.

Predictions were made of the secondary, two-dimensional (2-D) structures and side-chain solvent accessibilities of the light (L) chains of the clostridial neurotoxins (botulinum neurotoxin serotypes A-G and tetanus neurotoxin). An artificial neural network was used to make these predictions from a multiple alignment of their primary structures and was the approach used in making successful predictions for the C-fragments of these neurotoxins (Lebeda et al., J. Prot. Chem., 17:311, 1998). We also exploited the fact that the L-chains are Zn-dependent proteases. Although no other metalloproteases were found to be sequentially homologous to these neurotoxin L-chains, a sequence clustering algorithm showed that several bacterially derived Zn-dependent proteases, including thermolysin, were the most similar. A 2-D structure topology map for the type A L-chain was constructed by using thermolysin as a design template. As in thermolysin, the region containing the Zn-binding sequence motif, which is part of the active site in these neurotoxins, was predicted to be minimally solvent accessible. On the other hand, the locations of residues with highly exposed side chains were predicted to occur in non-periodic structure elements. Together, these 2-D structure and solvent accessibility predictions can be used to identify important solvent-exposed regions of the L-chain. These regions may include sites that interact with residues of the neurotoxin heavy chain, sites that bind to vesicle-docking substrates or sites that form antibody epitopes.

The topography of the closed-state membrane-associated, colicin E1 channel domain was examined using depth-dependent fluorescence quenching to determine the membrane location of various single Trp residues introduced into the sequence by site-directed mutagenesis. We have extended previous studies (Palmer, L. R., and Merrill, A. R. (1994) J. Biol. Chem. 269, 4187-4193) with additional single Trp residues in the helix 8/9 region, and with an additional quencher located in the polar region of the membrane to detect shallowly located Trp residues. Quenching data for seven single Trp mutants examined in the previous study, but without the shallow quencher, confirmed the previously reported depths. Mutants containing single Trp at residues 355, 460, or 507 were found to be more shallowly located than those at 404, 443, 484, or 495. In addition, analysis of fluorescence in the presence of the shallow quencher eliminated the possibility that there is a predominant population of these residues residing near the membrane-aqueous interface. The fluorescence quenching of three new single Trp at residues 478, 492, or 499 introduced into the channel domain was also evaluated. These residues were found at either medium or deep locations in the bilayer. Of special interest was the position of the Trp at residue 492 (W492), which is within the loop region connecting hydrophobic helices 8 and 9. If helices 8 and 9 were fully transmembraneous, then the predicted W492 location would have been shallow. Instead the quenching pattern demonstrated W492 to be deeply embedded in the lipid bilayer. We also studied the effect of altering bilayer width on protein conformation. Membrane width had little effect on most residues, but Trp at residues 478 and 507 were located more shallowly in thin bilayers. We also examined the effect of bilayer width on the position of Cys 505 labeled with bimane, an environmentally sensitive fluorophore. As the membrane width was decreased, C505-bimane shifted into a more nonpolar environment, as judged by fluorescence emission lambda max and quenching. Models for the conformation of helices 8/9 and the effect of membrane width on these helices are considered. We conclude that helices 8 and 9 probably do not adopt a fully transmembraneous state under the conditions examined in this report.

Clostridium perfringens epsilon-toxin bound to the Madin Darby canine kidney (MDCK) cells and aggregated. The complex of the toxin was formed in a dose- and a time-dependent manner. The formation of the complex increased with a decrease in viable counts of MDCK cells and with increasing K+ release from the cells. The inactivated toxin heated at 100 degrees C did not aggregate under the condition. In addition, the prototoxin dose-dependently bound to the cells, but did not form the complex. Incubation of the toxin with MDCK cell membranes also showed the formation of the complex, but that with membrane preparations prepared from Vero cells or sheep erythrocytes, which are insensitive for the toxin, showed no formation of the complex. Incubation of the toxin with mouse brain homogenates resulted in formation of the complex, but that with brain homogenates heated at 80 degrees C or mouse liver homogenates showed no formation of the complex. These observations show that the complex formation of epsilon-toxin is essential for toxicity of the toxin.