Phalloidin reduces the release of inorganic phosphate during actin polymerization

Phalloidin, an actin-filament stabilizing peptide from Amanita phalloides, did not inhibit ATP hydrolysis during actin polymerization but strongly retarded the release of the hydrolysis product Pi. Thus, the lifetime of the intermediate F-actin-ADP-Pi is significantly increased by phalloidin. The results suggest a close correlation between filament stability and F-actin-ADP-Pi intermediates.     

The insensitivity of mushroom nuclear RNA polymerase activity to inhibiton by amatoxins

Nuclear fractions isolated from Amanita phalloides, Amanita muscaria and Agaricus bisporus were subjected to in vitro RNA synthesis assays in the presence of various concentrations of amatoxins. The mushroom nuclei were highly insensitive to inhibition by amatoxin when compared to assays of nuclear fractions isolated from the Oömycete fungus, Achlya ambisexualis and from rabbit brain.Abbreviations DTT dithiothreitol - PMSF phenylmethyl sulfonyl fluoride - MES 2[N-morpholino] ethane sulfonic acid Paper no. 1-78     

长白山鹅膏菌肽类毒素的HPLC分析

采用HPLC法对长白山地区分布的10种鹅膏菌中的-鹅膏毒肽(-amanitin)、鹅膏毒肽(-amanitin)和鬼笔毒肽(phalloidin)3种毒素的含量进行了测定。结果表明:白鹅膏(A.verna)和鳞柄白鹅膏(A.virsa)中含有-amanitin和-amanitin两种毒素,二者-amanitin的含量分别为 1861.85g/g和2477.02g/g,均高于欧洲产毒鹅膏(A.phalloides)中的含量(1607g/g)而接近灰花纹鹅膏Amanita fuliginea中的量(2633.80g/g)。毒鹅膏A.phalloides中含有3种毒素,并且菌蕾中的含量高于成熟子实体,尤其菌蕾中Phalloidin的含量(1113.35g/g)是灰花纹鹅膏成熟子实体中(432.5g/g)的3倍。     

长白山鹅膏菌肽类毒素的HPLC分析*

采用HPLC法对长白山地区分布的10种鹅膏菌中的-鹅膏毒肽(-amanitin)、鹅膏毒肽(-amanitin)和鬼笔毒肽(phalloidin)3种毒素的含量进行了测定。结果表明:白鹅膏(A.verna)和鳞柄白鹅膏(A.virsa)中含有-amanitin和-amanitin两种毒素,二者-amanitin的含量分别为 1861.85g/g和2477.02g/g,均高于欧洲产毒鹅膏(A.phalloides)中的含量(1607g/g)而接近灰花纹鹅膏Amanita fuliginea中的量(2633.80g/g)。毒鹅膏A.phalloides中含有3种毒素,并且菌蕾中的含量高于成熟子实体,尤其菌蕾中Phalloidin的含量(1113.35g/g)是灰花纹鹅膏成熟子实体中(432.5g/g)的3倍。     

Influence of phalloidin on both the nucleation and the elongation phase of actin polymerization

Phalloidin, a heptapeptide from the mushroom Amanita phalloides, increased the velocity of actin polymerization, but slightly decreased the velocity of elongation (polymerization onto sonicated F-actin). A plot of log polymerization velocity vs. log actin concentration was less steep in the presence of phalloidin than in its absence, suggesting that the filament nucleus is smaller in the presence of phalloidin than in its absence.     

The effect of phalloidin and its nontoxic derivative dethiophalloidin on morphological transformation of mitochondria

As the in vitro experiments showed, the process of osmotic swelling of isolated liver mitochondria was inhibited by phalloidine produced by mushrooms Amanita phalloides. The degree of the effect pronounced depended both on the heptapeptide concentration and duration of the action on mitochondria. Mitochondrial contraction caused by ATP adding was also inhibited. The control experiments performed using dethiophalloidine as a nontoxic derivate showed the absence of similar effect. The data obtained point to functional significance of the actin-like protein as a factor capable of forming a continuous primembrane system connected with the integral components and controlling the volume of mitochondria themselves.     

Amatoxins in wood-rotting Galerina marginata

Amatoxins, bicyclic octapeptide derivatives responsible for severe hepatic failure, are present in several Basidiomycota species belonging to four genera, i.e. Amanita, Conocybe, Galerina and Lepiota. DNA studies for G. autumnalis, G. marginata, G. oregonensis, G. unicolor and G. venenata (section Naucoriopsis) determined that these species are the same, supporting the concept of Galerina marginata complex. These mostly lignicolous species are designated as white-rot fungi having a broad host range and capable of degrading both hardwoods and softwoods. Twenty-seven G. marginata basidiomes taken from different sites and hosts (three sets) as well as 17 A. phalloides specimens (three sets) were collected in French locations. The 44 basidiomes were examined for amatoxins and phallotoxins using high-performance liquid chromatography. Toxinological data for the wood-rotting G. marginata and the ectomycorrhizal A. phalloides species were compared and statistically analyzed. The acidic and neutral phallotoxins were not detected in any G. marginata specimen, whereas the acidic (β-Ama) and neutral (α-Ama and γ-Ama) amanitins were found in all basidiomes from either Angiosperms or Gymnosperms hosts. The G. marginata amatoxin content varied from 78.17 to 243.61 μg.mg(-1) of fresh weight and was elevated significantly in one set out of three. The amanitin amounts from certain Galerina specimens were higher than those from some A. phalloides basidiomes. Relationship between the amanitin distribution and the chemical composition of substrate was underlined and statistically validated for the white-rot G. marginata. Changes in nutritional components from decayed host due to enzymatic systems and genetic factors as well as environmental conditions seem to play a determinant role in the amanitin profile. Variability noticed in the amanitin distribution for the white-rot G. marginata basidiomes was not observed for the ectomycorrhizal A. phalloides specimens.     

Antamanide, a derivative of Amanita phalloides, is a novel inhibitor of the mitochondrial permeability transition pore

Antamanide is a cyclic decapeptide derived from the fungus Amanita phalloides. Here we show that antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by antamanide is not additive with the cyclophilin D-binding drug cyclosporin A, (ii) the inhibitory action of antamanide on the pore requires phosphate, as previously shown for cyclosporin A; (iii) antamanide is ineffective in mitochondria or cells derived from cyclophilin D null animals, and (iv) abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability transition pore inhibition by antamanide needs two critical residues in the peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on the pore in a cyclophilin D-independent fashion. Antamanide also abrogates mitochondrial depolarization and the ensuing cell death caused by two well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal peptide. Our findings have implications for the comprehension of cyclophilin D activity on the permeability transition pore and for the development of novel pore-targeting drugs exploitable as cell death inhibitors.     

Mushroom poisoning

In the research we included a total of 207 subjects with the dismissal diagnosis of "mycetismus", who were treated at the Department of Infectious Diseases, General Hospital Osijek, during the 1983-1992 period. 32 of them were children. There were 44.93% of men, 39.61% of women and 15.45% of children. The latent time > 6 hours was determined in 51 (25%) and < 6 hours in 75% of subjects. In 156 of patients with the latent time > 6 hours, "false" poisoning occured, while 51 patients experienced real mushroom toxins poisoning. At the admission to the hospital, in patients with the latent time > 6 hours, a pathological PT (protrombine time) was established only in women, leukocytosis in both women and children, increased concentration of GGT (gamma-glutamin-transferase) in men, increased AST (aspartate-aminotransferase) and ALT (alanin-aminotransferase) only in women, and increased urea in both women and children. After 24 hours, control measuring established high values of AST and ALT extended PT uremia and exalted amount of ammonia in blood in 11 of patients (2 men, 7 women and 2 children). They had severe liver and kidney damage, the most probably caused by Amanita phalloides toxins. The latent time lasted 9 to 13 hours. Of the 11 above mentioned patients, 2 women, aged 74 and 43, and one girl, aged 6, died. No pathological laboratory parameters were established in 40 of subjects with the latent time of 6 and more hours, and the disease manifested through vomiting and diarrhea that lasted for several days. These subjects most probably suffered from mushroom toxins poisoning. Mushroom toxins irritate the mucuous membrane of the gastrointestinal tract, and there are many such poisonous mushrooms. There were no mortalities in this group of subjects.     

Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms.

This review gives a comprehensive account of the molecular toxicology of the bicyclic peptides obtained from the poisonous mushrooms of the genus Amanita. The discussion of the biochemical events will be preceded by a consideration of the chemistry of the toxic peptides. The structural features essential for biological activities of both the amatoxins and the phallotoxins will be discussed, also including the most important analytical data. Similar consideration will be given to antamanide, a cyclic peptide, which counteracts phalloidin. In addition, the phallolysins, three cytolytic proteins from Amanita phalloides will be discussed. The report on the biological activity of the amatoxins will deal with the sensitivity of the different RNA-polymerases towards the toxins and with their action on various cell types. Consideration will also be given to systems in which alpha-amanitin was used and can be used as a molecular tool; in the past, many investigators used the inhibitor in molecular biology, genetics, and even in physiological research. As for the phallotoxins, discussion of the affinity of these toxins for actin is provied. Further discussion attempts to understand the course of intoxication by filling in the gap between the first molecular event, formation of microfilaments, and the various lesions in hepatocytes during the intoxication.     

Ribosomal biosynthesis of α-amanitin in Galerina marginata

Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerinamarginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerinahybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G.marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear.     

Artificial and Bioartificial Liver Support

The liver is a complex organ with various vital functions in synthesis, detoxification and regulation; its failure therefore constitutes a life threatening condition1. Liver failure (LF) can either occur without preceding liver disease (acute liver failure, ALF), usually caused either by intoxication (Amanita phalloides, acetaminophen, methylendioxymethamphteamine) or as acute decompensation of chronic liver-related illness (acute-on-chronic liver failure, AoCLF). In both cases, its symptoms include icterus, hepatic encephalopathy and impairment of coagulation status and may result in multi organ failure. Exceptionally, liver failure may also be triggered by certain diseases (Budd-Chiari-syndrome, Morbus Wilson) or pregnancy. The only long-term therapy in most cases is orthotopic liver transplantation, unless the liver is able to regenerate. Many patients, especially those who are not listed for high urgency transplantation, may not survive until a suitable donor organ is available, since donor organs are rare. In other cases, contraindications do not permit liver transplantation. For these indications, extracorporeal liver assist devices have been developed in order to either bridge the patient to transplantation or temporarily support the failing organ until it is able to regenerate.     

我国28种鹅膏菌主要肽类毒素的检测分析*

利用高效液相色谱(HPLC)技术对产于我国的28种鹅膏菌的主要肽类毒素(鹅膏毒肽和鬼笔毒肽)进行了检测分析,并和采于欧洲(德国)的毒鹅膏Amanita phalloides作对照,结果表明,3种东亚所特有的鹅膏菌(灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种)和欧洲毒鹅膏所含毒素种类多、含量高,其子实体菌盖部位主要毒素总量分别达到12583.7μg/g、8152.6μg/g、1058.2μg/g、7456.2μg/g干重子实体,这4种鹅膏菌可称之为剧毒鹅膏菌。其它25种鹅膏菌中有10种检测出含有微量鹅膏毒肽,含量在19.5μg/g-151.2μg/g之间。在4种剧毒鹅膏菌中,子实体组织部位不同,毒素含量以及鹅膏毒肽和鬼笔毒肽在其中的分布也不一样,菌盖中的毒素含量最高,菌柄的毒素含量次之,菌托中的毒素含量最低;对于灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种,无论在菌盖、菌柄和菌托中,鹅膏毒肽类毒素的含量都高于鬼笔毒肽类毒素,尤其以α-amanitin的相对含量最高;而在欧洲毒鹅膏中,菌盖、菌柄和菌托中都以鬼笔毒肽为主,尤其以phallacidin的相对含量最高,并且从菌盖至菌柄到菌托,鬼笔毒肽的相对含量依次增加。     

中国鹅膏菌属(担子菌)的物种多样性

鹅膏菌属（Ａｍａｎｉｔａ）是一个近世界性广布的大属,全球已被描述而又被承认的有近５００种。在文献中,我国此属已记载约２００种。然而,许多种都是原初描述于欧洲或北美的种类。近来的研究表明,东亚的鹅膏菌独特且有其自身的分布范围。东亚的有些种虽与产于欧洲或北美的某些种相似,但仔细的野外观察,详尽的形态解剖学和分子进化生物学研究结果表明,东亚的鹅膏菌是独立的分类群。在欧洲,人们有采集著名食菌恺撒鹅膏菌的习     

Suppression of secondary hyperparathyroidism in children with chronic renal failure by high dose phosphate binders: calcium carbonate versus aluminium hydroxide.

Secondary hyperparathyroidism was suppressed over a period of one year in 12 children with chronic renal failure by using a regimen of mild dietary phosphate restriction and high dose phosphate binders. The patients were randomised to receive either aluminium hydroxide or calcium carbonate by mouth for six months and then crossed over to the other medication. Vitamin D (dihydrotachysterol) dosage was unchanged. Serum parathyroid hormone concentrations were reduced to within the normal range, urinary cyclic adenosine monophosphate values fell, plasma phosphate concentrations decreased, and the theoretical renal phosphate threshold increased significantly. Transiliac bone biopsy findings improved in four patients with adequate suppression of parathyroid hormone concentrations, deteriorated in two patients who were not compliant, and did not change in five patients in whom initial bone disease was mild. Growth velocity improved significantly. There was no difference in the clinical response, biochemical changes, or incidence of complications during treatment with the two agents. In view of the risk of aluminium toxicity the use of high dose calcium carbonate with dietary phosphate restriction and vitamin D supplementation is recommended in the control of secondary hyperparathyroidism in children with chronic renal failure.     

Distribution of the amatoxins and phallotoxins in Amanita phalloides. Influence of the tissues and the collection site

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.     

Interaction of actin with phalloidin: polymerization and stabilization of F-actin.

The cyclic peptide phalloidin, one of the toxic components of Amanita phalloides prevented the drop of viscosity of F-actin solutions after the addition of 0.6 M KI and inhibited the ATP splitting of F-actin during sonic vibration. The data concerning ATP splitting are consistent with the assumption (a) that only 1 out of every 3 actin units of the filaments needs to be combined with phalloidin in order to suppress the contribution of these 3 actins to the ATPase activity of the filament and (b) that all actin units of the filaments can combine with phalloidin with a very high affinity. -halloidin did not only stabilize the actin-actin bonds in the F-actin structure but it also increased the rate of polymerization of G-actin to F-actin. The ability of F-actin to activate myosin ATPase was not affected by phalloidin. The tropomyosin-troponin complex did not prevent the stabilizing effect of phalloidin on the F-actin structure.     

Parathyroid hormone in urinary stone patients

To evaluate the role of parathyroids in calculus disease, the parathyroid hormone levels were determined in 22 control subjects and 42 stone (14 with bladder stone and 28 with kidney stone) patients. Serum calcium, inorganic phosphate, alkaline phosphatase and parathyroid hormone and urinary excretion of calcium and inorganic phosphate were determined. It was found that normocalcemic and normocalciuric stone patients had slightly higher levelsss of parathyroid hormone (irrespective of the site of the stone) and the difference was not statistically significant as compared with control subjects although some of the patients with calculus disease were hyperparathyroid. Serum alkaline phosphatase was increased while there was an increase in urinary calcium excretion in kidney stone patients and oxalate in all patients as compared with control subjects. The increase in inorganic phosphate was, however, not different from the control subjects. The subclinical hyperparathyroidism and stone formation in these patients are not correlated.     

Selected indicators of calcium-phosphate metabolism in patients with diabetes mellitus

The changes in blood serum concentrations of calcium, magnesium, inorganic phosphate, total activity of alkaline phosphatase and the activity of its bone fraction, as well as urinary excretion of calcium, phosphate, hydroxyproline and oxalate have been measured in 31 patients with insulin-dependent (type I) diabetes, in 31 patients with non-insulin-dependent (type II) diabetes and in 29 healthy subjects in the condition of low-calcium diet. The elevated urinary excretion of calcium, phosphate, hydroxyproline and oxalate, lowered blood serum level of magnesium, and increased total and bone fraction activities of alkaline phosphatase were found in diabetic patients. The urinary excretion of calcium and hydroxyproline, and the activity of bone fraction alkaline phosphatase were significantly higher in patients with type II diabetes than in those with type I diabetes. It was concluded that there is a significant relation between the state of metabolic normalization of diabetes and the degree of biochemical aberrations concerning calcium-phosphate metabolism.     

鹅膏菌中毒小鼠解毒药物的筛选

考察了几种中药对鳞柄白鹅膏中毒小鼠的解毒作用。鳞柄白鹅膏为极毒真菌,当其干燥子实体水溶液质量浓度>10mg/mL时,小鼠全部死亡,质量浓度为10mg/mL时,小鼠在9 h内全部死亡,选择这一浓度使小鼠中毒,使用所选中药对小鼠进行解毒试验。结果表明:水飞蓟(7.8 g/kg)、绿豆(3.9 g/kg)及甘草(1.3 g/kg)均能延迟小鼠的死亡时间,但与对照组相比差异不显著。灵芝(1.95 g/kg)对小鼠的死亡时间有明显的延迟作用,并与对照组相比差异显著(P<0.05)。同时使用灵芝剂量为首次解毒试验剂量的100倍水煎液(195 g/kg)和300倍水煎液(585 g/kg),以及按原料剂量为195 g/kg提取其中三萜及多糖用于对鹅膏中毒小鼠的解毒试验。结果表明:试验组小鼠的死亡时间均有延迟,其中195 g/kg剂量灵芝组和灵芝三萜试验组与对照组相比差异显著(P<0.05)。