amanitins and phallacidin

Topics: Actins; Amanitins; Animals; Bisbenzimidazole; DNA; Embryo, Nonmammalian; Fixatives; Fluorescent Antibody Technique; Histocytochemistry; Micromanipulation; Microscopy, Fluorescence; Microtomy; Ovum; Peptides, Cyclic; Rhodamines; Sea Urchins; Staining and Labeling

Amanita fuligineoides, a lethal mushroom discovered in China, contains abundant cyclic peptide toxins that can cause fatal poisoning. However, the MSDIN gene family encoding for these cyclic peptides in A. fuligineoides has not been systematically studied. In this research, the transcriptome sequencing of A. fuligineoides was performed and its MSDIN family members were analyzed. A total of 4.41 Gb data containing 30833 unigenes was obtained; sequence alignments throughout several databases were done to obtain their functional annotations. Based on these annotations, MSDIN genes were found and verified by RT-PCR. A total of 29 different core peptides were obtained: 3 toxin genes, encoding β-amanitin (β-AMA), phalloidin (PHD), and phallacidin (PCD), and 26 genes encoding unknown cyclic peptides, 20 of which are reported for the first time and may encode for novel cyclic peptides. Analysis of the predicted precursor peptides indicated that octocyclic peptides were the main MSDIN peptides synthesized by A. fuligineoides, accounting for the 45%. A phylogenetic analysis suggested that studied precursor peptides could be clustered into 7 clades, which might represent different functionalities. Results suggested that A. fuligineoides might have a strong capacity to synthesize cyclopeptides, laying the foundation for their excavation and utilization.

Topics: Alpha-Amanitin; Amanita; Amanitins; Amino Acid Sequence; China; Gene Expression Profiling; Peptides, Cyclic; Sequence Alignment; Toxins, Biological; Transcriptome

In this study, the toxicology of A. exitialis, a lethal mushroom found in China, and the toxicokinetics of peptide toxins contained in it were evaluated. Beagles were fed A. exitialis powder (20 or 60 mg/kg) in starch capsules, after which they were assessed for signs of toxicity, as well as biochemical and pathological changes. Ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to assay the peptide toxins. The total peptide toxins in A. exitialis was 3482.6 ± 124.94 mg/kg. The beagles showed signs of toxicity, such as vomiting and diarrhea, at 12-48 h following ingestion of A. exitialis. Furthermore, alanine transaminase and aspartate transaminase levels in plasma, as well as prothrombin time and activated partial thromboplastin time peaked at 36 h post A. exitialis ingestion. Furthermore, total bilirubin and alkaline phosphatase levels peaked at 48 h after A. exitialis ingestion. Three dogs that were administered 60 mg/kg A. exitialis died at 24-72 h after ingesting the capsules. Additionally, liver histopathological examinations showed hemorrhagic necrosis of hepatocytes. α-Amanitin, β-amanitin, and phallacidin were rapidly absorbed and eliminated from plasma after A. exitialis was ingested. A long latency period (12-24 h post A. exitialis ingestion) was observed in the dogs before the onset of gastrointestinal symptoms. There was acute liver damage thereafter. Gastric lavage and enhanced plasma clearance methods such as hemodialysis, hemoperfusion, or plasma exchange may be ineffective in removing amatoxins from blood at 12 h post A. exitialis ingestion. Enhanced excretion of amatoxins in urine could be effective within 2 days after ingestion of A. exitialis because amatoxins in 0-2 d urine accounted for more than 90% of the total urine excretion.

Topics: Alanine Transaminase; Amanita; Amanitins; Animals; Aspartate Aminotransferases; Dogs; Fungal Proteins; Liver Diseases; Male; Mushroom Poisoning; Partial Thromboplastin Time; Peptides, Cyclic; Prothrombin Time; Toxicokinetics

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, β-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, β-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

Topics: Alpha-Amanitin; Amanita; Amanitins; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Forests; Fruiting Bodies, Fungal; Humans; Mushroom Poisoning; Mycelium; Peptides, Cyclic; Phalloidine; Species Specificity; Spectrophotometry, Ultraviolet; Spores, Fungal; Turkey

Amanita exitialis is a lethal mushroom that was first discovered in Guangdong Province, China. The high content of amanitin in its basidiocarps makes it lethal to humans. To comprehensively characterize the A. exitialis transcriptome and analyze the Amanita toxins as well as their related gene family, transcriptome sequencing of A. exitialis was performed using Illumina HiSeq 2000 technology. A total of 25,563,688 clean reads were collected and assembled into 62,137 cDNA contigs with an average length of 481 bp and N50 length of 788 bp. A total of 27,826 proteins and 39,661 unigenes were identified among the assembled contigs. All of the unigenes were classified into 166 functional categories for understanding the gene functions and regulation pathways. The genes contributing to toxic peptide biosynthesis were analyzed. From this set, eleven gene sequences encoding the toxins or related cyclic peptides were discovered in the transcriptome. Three of these sequences matched the peptide toxins α-amanitin, β-amanitin, and phallacidin, while others matched amanexitide and seven matched unknown peptides. All of the genes encoding peptide toxins were confirmed by polymerase chain reaction (PCR) in A. exitialis, and the phylogenetic relationships among these proprotein sequences were discussed. The gene polymorphism and degeneracy of the toxin encoding sequences were found and analyzed. This study provides the first primary transcriptome of A. exitialis, which provided comprehensive gene expression information on the lethal amanitas at the transcriptional level, and could lay a strong foundation for functional genomics studies in those fungi.

Topics: Alpha-Amanitin; Amanita; Amanitins; Amino Acid Sequence; Base Sequence; Fruiting Bodies, Fungal; Fungal Proteins; Molecular Sequence Annotation; Molecular Sequence Data; Peptides, Cyclic; Phylogeny; Polymorphism, Genetic; Sequence Analysis, DNA; Transcriptome

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and β-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.

Topics: Amanita; Amanitins; Chromatography, Liquid; Humans; Mass Spectrometry; Mushroom Poisoning; Peptides, Cyclic; Phalloidine; Poisons

The identification of toxic oligopeptides employing CE-ESI-MS is presented. The analytes studied ama- and phallotoxins are of significant forensic interest because over 90% of the lethal cases of fungus poisoning in man are caused by species of Amanita which contain these toxins. A CE method was developed to separate the toxins alpha-, beta- and gamma-amanitin, phalloidin and phallacidin. Their fragmentation patterns in MS(n) experiments were investigated in the positive and in the negative ion mode, also the influence of the sheath liquid mixture of the used interface on the S/N. Method validation included the determination of the LOD and the repeatability of the migration time and peak area for both detection modes. With the optimized method LODs of 13-79 ng/mL (17-87 nmol/L) were reached. The CE-MS procedure was successfully applied to the identification of ama- and phallotoxins in extracts of air-dried mushroom samples.

Topics: Amanita; Amanitins; Electrophoresis, Capillary; Humans; Molecular Structure; Mushroom Poisoning; Mycotoxins; Oligopeptides; Peptides, Cyclic; Phalloidine; Spectrometry, Mass, Electrospray Ionization

The toxin composition of 25 Amanita phalloides carpophores collected from three sites in Franche-Comté (France) differing in their geological and pedological characteristics was determined and the factors involved in the variations of the toxin concentration in the tissues were identified. The concentrations of the main amatoxins (beta-amanitin, alpha-amanitin, gamma-amanitin) and phallotoxins (phallacidin, phallisacin, phalloidin, phallisin, phalloin) in the six tissues constituting the carpophore, i.e. the cap (C), gills (G), ring (R), stipe (S), bulb (B) and volva (V) were evaluated by using high-performance liquid chromatography. The results analysed statistically showed that the toxin concentrations were tissue dependent, leading to classification of the tissues into two groups (B, V) and (C, G, R, S). The (B, V) group was distinguished by high amounts of phalloidin, phallisin and phallisacin, and the (C, G, R, S) group by the predominance of the amatoxins. The characteristics of the soil of the collection site also affected the toxin concentrations; however, this effect differed from one site to another and was not similar for all the tissues. Finally, the mean toxin profile in the carpophores from the three sites was evaluated. This study underscores the fact that environmental factors and mainly the soil type clearly have an effect on the toxin composition of A. phalloides carpophores.

Topics: Alkaloids; Amanita; Amanitins; Environment; France; Hydrogen-Ion Concentration; Peptides, Cyclic; Phalloidine; Soil

alpha-Thrombin interacts with confluent bovine pulmonary artery endothelial cells to produce two types of actin microfilament rearrangements: (1) The loss of cortical actin correlates with interruption of barrier integrity, evident from increased permeability to 125I-albumin; and (2) an increase in actin stress fibers results in greater adherence of the cells to their extracellular substrate. Use of phallatoxin compounds that stabilize actin filaments and prevent their depolymerization prevents both the cytoarchitectural and functional changes resulting from thrombin challenge.

Topics: Actins; Amanitins; Animals; Cattle; Cell Adhesion; Cell Division; Cell Survival; Cells, Cultured; Endothelium, Vascular; Fluorescent Dyes; Microscopy, Fluorescence; Peptides, Cyclic; Thrombin

The conjugates beta-amanitin-concanavalin A and phallacidin concanavalin A were tested for direct cytotoxicity on L1210 lymphocytic leukemia cells by a combined in vitro-in vivo bioassay. Both conjugates exerted strong direct cytotoxicity on the tumour cells.

Topics: Amanitins; Animals; Cell Survival; Concanavalin A; Cytotoxins; Immunotoxins; Leukemia L1210; Peptides, Cyclic; Tumor Cells, Cultured

High titre antiserum was obtained by immunization of rabbits with phalacidine-concanavaline A. Specific antiphalacidine antibodies were isolated from this serum on immobilized phalacidine. It was established by testing with antirabbit globulin sera that these antibodies were of IgG class only. IgG fraction was isolated from the immune serum by ion-exchange chromatography, but specific antibodies, which represented 8% of IgG of the serum, were isolated from it by affinity chromatography. The antibodies isolated by affinity, were tested against other phalatoxines and amotoxines. The results showed that they reacted with phalatoxines, but not with amatoxines and could be used in tests for quantitative determination of phalatoxines in mushroom extracts and body fluids.

Topics: Amanitins; Animals; Antibodies; Antibody Specificity; Chromatography, Affinity; Chromatography, Gel; Concanavalin A; Immunization; Immunodiffusion; Male; Peptides, Cyclic; Rabbits

Filaments about 6-7 nm in diameter were seen associated with germ cell intercellular bridges in detergent-permeabilized cells treated with tannic acid. Approximately 40-50 filaments were present subjacent to the bridge density. Filaments encircled the bridge channel in a manner similar to contractile ring actin filaments of dividing cells. NBD-phallacidin and myosin S-1 subfragments were employed to demonstrate that the filaments observed at intercellular bridges are actin. Intratesticular injection of a single dose of cytochalasin D, a specific inhibitor of actin filaments, caused certain intercellular bridges of spermatids to open within 3 hr after injection, leading to the production of symplasts. During bridge opening, remnants of bridge densities were gradually incorporated into the lateral aspect of the plasma membrane of the symplast. Thus actin, present in bridge structures, appeared to participate in maintaining certain intercellular bridges. A model of intercellular bridge structure is presented.

Topics: Actins; Amanitins; Animals; Cytochalasin D; Cytochalasins; Epithelium; Intercellular Junctions; Male; Microscopy, Electron; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Models, Biological; Peptides, Cyclic; Rats; Rats, Inbred Strains; Sciuridae; Spermatids; Spermatocytes; Testis

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.

Topics: Actins; Amanitins; Animals; Fertilization; Microscopy, Fluorescence; Ovum; Peptides, Cyclic; Polymers; Protein Binding; Sea Urchins; Time Factors

Primary cultures of rat hepatocytes were used for assaying several drugs not previously known for inhibiting the transport of phalloidin. In order to have 50% inhibition (IC50) of the entrance of a tritiated phallotoxin derivative ([3H]demethylphalloin, 1 microM) from the medium into the cells the following concentrations (microM) of the various inhibitors were determined: cyclolinopeptide (0.5), Nocloprost (5.0), Nileprost (7.0), beta-estradiol (42), Verapamil (70). For comparison, the corresponding IC50 values of some known antagonists of phalloidin toxicity were determined by the same method. Moreover, we studied several natural and synthetic phallotoxins and alpha-amanitin for their ability to displace [3H]demethylphalloin from the transporting system. Lineweaver-Burk plots made it obvious that two groups of inhibitors exist. Competitive inhibitors are, for example, antamanide, beta-estradiol, silybin, Nileprost, taurocholate, and the cyclic somatostatin analog cyclo[Phe-Thr-Lys-Trp-Phe-D-Pro], whereas Verapamil and monensin inhibit phallotoxin uptake in a non-competitive way. Considering the very different chemical features of the competitive inhibitors, we tentatively conclude that the phallotoxin transport system selects compounds not on the basis of their chemical features, but rather their physical properties. The physical properties of a typical substrate are low molecular mass, lipophilic nature, and, possibly the presence of rigid ring structures. Negative charges accelerate the transport of a substrate, while positive charges have the opposite effect. The phalloidin-transporting system may represent part of a hepatic equipment which clears portal blood from, for example, bile acids, lipophilic hormones, or xenobiotics. By chance, the transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.

Topics: Amanitins; Animals; Binding, Competitive; Biological Transport; Cells, Cultured; Cholic Acids; Epoprostenol; Estradiol; Kinetics; Liver; Monensin; Oligopeptides; Peptides, Cyclic; Phalloidine; Prostaglandins F, Synthetic; Rats; Silybin; Silymarin; Somatostatin; Taurocholic Acid; Verapamil

Topics: Actins; Amanitins; Animals; Cytoskeleton; Incisor; Intermediate Filaments; Microscopy, Electron; Microtubules; Myosin Subfragments; Odontoblasts; Peptides, Cyclic; Rats

The effect of an antidiuretic hormone (ADH, vasopressin) on the microfilament system of the toad urinary bladder lumenal epithelium was investigated using NBD-phallacidin (NBD-ph). The latter material is a specific fluorescent label for F actin. In the presence of an osmotic gradient, both ADH and cyclic adenosine monophosphate (cAMP) appear to induce the polymerization of monomeric actin into F actin-containing microfilaments. The latter may then be involved in the morphological changes, including the formation of lateral intercellular lakes, associated with the typical hydroosmotic response.

Topics: Actins; Amanitins; Animals; Arginine Vasopressin; Bufo marinus; Cyclic AMP; Cytoskeleton; Epithelium; Female; Microscopy, Fluorescence; Peptides, Cyclic; Thionucleotides; Urinary Bladder

The spatial distribution of cytoplasmic actin in endoplasmic drops as well as in plasmodial strands can be demonstrated in cryosections by fluorescently labelled phallotoxins and actin antibodies. Our results on cryosections show an identical fibrillar actin distribution as revealed in semithin sections after conventional fixation and embedding. Thus, it is now possible to apply immunocytochemical analysis to any and all plasmodial stages with or without prior fixation and without using extraction procedures. Consequentially the loss of soluble compounds during processing is avoided. The most protective pretreatment of the living specimens before freezing is a 15 min incubation in 1.5 M sucrose containing 50 mM KCl, 10 mM EGTA and 10 mM PIPES buffer, pH 7.0, at 4 degrees C.

Topics: Actins; Amanitins; Cytoskeleton; Fixatives; Fluorescent Antibody Technique; Freezing; Peptides, Cyclic; Physarum

This study demonstrates a novel feature of PMA, its ability to suppress chemokinetic polarization and locomotion of tumor cells. Walker carcinosarcoma cells exhibit two distinct types of polarization and locomotion, i.e. spontaneous polarization characterized by ruffles at the front and stimulated polarization and locomotion in response to the microtubule-disassembling agents colchicine, vinblastine and nocodazole, which are characterized by blebbing at the front. The tumor promotor phorbol myristate acetate (PMA), but not phorbol, was found to suppress both types of polarization and random locomotion at concentrations between 10(-8) and 10(-6)M. The effect of 10(-6)M PMA was virtually complete within 5 min. Inhibition of locomotion was due to both a reduction in the speed of migrating cells and the proportion of migrating cells. Changes in shape and chemokinesis of Walker carcinosarcoma cells were associated with alterations in the relative amount and the topographical distribution of F-actin as determined by NBD-phallacidin binding. Suppression by PMA was associated with loss of the polar topographical distribution of F-actin visualized by NBD-phallacidin binding. In the presence of PMA, the relative amount of F-actin was higher than in unstimulated controls and lower in cells exposed to microtubule-disassembling agents.

Topics: Actin Cytoskeleton; Actins; Amanitins; Animals; Carcinoma 256, Walker; Cell Adhesion; Cell Movement; Colchicine; Cytoskeleton; Microtubules; Peptides, Cyclic; Phorbols; Rats; Tetradecanoylphorbol Acetate; Vinblastine

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X-100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.

Topics: Actins; Amanitins; Animals; Axons; Cells, Cultured; Chick Embryo; Cytoskeleton; Microscopy, Electron; Microtubules; Myosin Subfragments; Myosins; Neurons, Afferent; Peptide Fragments; Peptides, Cyclic

Topics: Acrosome; Actins; Amanitins; Ammonia; Animals; Calcimycin; Exocytosis; Fluorescent Dyes; Male; Peptides, Cyclic; Polymers; Spermatozoa; Starfish

Topics: Actins; Amanitins; Animals; Chromatography; Fluorescent Dyes; Macropodidae; Peptides, Cyclic; Spectrometry, Fluorescence