a-鹅膏毒肽和二羟鬼笔毒肽的制备和鉴定(英文)

-鹅膏毒(环)肽和二羟鬼笔毒(环)肽是剧毒的鹅膏菌和其它几种致死毒菌中由一些修饰氨基酸组成的环肽毒素。由于-鹅膏毒肽对真核生物的mRNA合成的专一性抑制和和二羟鬼笔毒肽对肌动蛋白的专一性束缚,因而它们在分子生物学和细胞学研究中具有重要应用,对其需求逐步增加。为此,作者使用了一种改良的毒素提取方法,以制备高效液相色谱从灰花纹鹅膏菌中分离制备-鹅膏毒肽和二羟鬼笔毒肽,并通过紫外吸收光谱和质谱进行鉴定,表明-鹅膏毒肽和二羟鬼笔毒肽的分离效果好,纯度高。本方法对其它毒菌中的-鹅膏毒肽和二羟鬼笔毒肽的分离制备具有同样的应用价值。     

α-鹅膏毒肽和二羟鬼笔毒肽的制备和鉴定

α-鹅膏毒(环)肽和二羟鬼笔毒(环)肽是剧毒的鹅膏菌和其它几种致死毒菌中由一些修饰氨基酸组成的环肽毒素.由于α-鹅膏毒肽对真核生物的mRNA合成的专一性抑制和和二羟鬼笔毒肽对肌动蛋白的专一性束缚,因而它们在分子生物学和细胞学研究中具有重要应用,对其需求逐步增加.为此,作者使用了一种改良的毒素提取方法,以制备高效液相色谱从灰花纹鹅膏菌中分离制备α-鹅膏毒肽和二羟鬼笔毒肽,并通过紫外吸收光谱和质谱进行鉴定,表明α-鹅膏毒肽和二羟鬼笔毒肽的分离效果好,纯度高.本方法对其它毒菌中的α-鹅膏毒肽和二羟鬼笔毒肽的分离制备具有同样的应用价值.     

云南楚雄致命鹅膏中环肽毒素的检测与分析

采用反相高效液相色谱法对采自云南楚雄双柏县的致命鹅膏在3个不同生长期中不同部位的6种环肽毒素含量进行了检测和分析。结果表明,致命鹅膏含有α-, β-鹅膏毒肽、羧基三羟鬼笔毒肽和羧基二羟鬼笔毒肽,未检出γ-鹅膏毒肽和二羟鬼笔毒肽。生长期毒素总量最高（9.3mg/g）、从成熟期（7.5mg/g）到衰老期（6.5mg/g）逐渐降低,但鬼笔毒肽的相对含量随着年龄增长而逐渐增加,鹅膏毒肽与鬼笔毒肽比值从生长期、成熟期到衰老期分别为2.6、1.4和0.9。在3个不同发育阶段中,4种毒素含量从菌盖、菌柄到菌托逐渐降低,而鬼笔毒肽的相对含量逐渐增加。α-鹅膏毒肽和β-鹅膏毒肽在生长期菌盖中含量最高,分别为7.4mg/g和3.1mg/g,而羧基三羟鬼笔毒肽和羧基二羟鬼笔毒肽在衰老期的菌盖中含量最高,分别为2.8mg/g和2.1mg/g。     

β-鹅膏毒肽的反相高效液相色谱分离纯化*

用反相高效液相色谱,以0.02mol/L醋酸铵-乙腈为流动相的梯度洗脱模式,在295nm吸收值的条件下,灰花纹鹅膏菌Amanitafuliginea的肽类毒素可以被成功的分离和纯化。单个肽类毒素的鉴定是用反相高效液相色谱和质谱同时进行。用这一方法可从灰花纹鹅膏菌中分离纯化出β-鹅膏毒肽(β-amanitin),产量可达到:1158靏/g(干重),产品纯度达98%以上,回收率为95.3%。β-鹅膏毒肽的分子量为919.3Da。这个方法可用于其它鹅膏菌肽类毒素的分离纯化。     

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。     

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.     

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。     

7种鹅膏菌属真菌肽类毒素的HPLC分析

利用HPLC法对长白山地区分布的7种鹅膏属真菌成熟子实体中的α鹅膏毒肽(αamanitin)、β鹅膏毒肽(βamanitin)和鬼笔毒肽(phalloidin)的含量进行了测定。结果表明:芥橙黄鹅膏(Amanitasubjunquillea)和橙黄鹅膏(Amanitaaff.citrina)中均含有3种毒素,其中芥橙黄鹅膏的α鹅膏毒肽含量为2395.91μg/g、β鹅膏毒肽含量为1653.75μg/g和鬼笔毒肽含量为405.26μg/g;橙黄鹅膏的分别为1121μg/g、4244μg/g和9442μg/g。芥橙黄鹅膏白色变种(Amanitasubjunquilleavar.abla)中含有β鹅膏毒肽,其含量为614.00μg/g。其他5种鹅膏中均未检测到上述3种毒素。     

三种鹅膏菌培养条件及所产肽类毒素的比较研究

以外生菌根菌鹅膏菌属三个种Amanita muscaria,A.pseudoporphyria和A.fritillaria为研究材料,以生长速率为评价指标,对其最适生长温度、pH值、光照、培养基、C及N源的利用等基本培养条件及所产肽类毒素进行了比较研究。研究结果表明,三种菌株最适生长温度有差异,A.pseudoporphyria和A.fritillaria的最适温度为28℃,A.muscaria的最适温度为22℃;A.muscaria菌丝体生长的pH值范围为5-7,另外两个菌株的pH值范围为3-6;24h光照、12h光暗交替和24h黑暗对鹅膏菌的生长速率影响不大;SPDM培养基和MMN培养基都适合三种菌株的生长,但对于A.muscaria来说PDM培养基更适合其生长。鹅膏菌能够利用比较广泛的C、N源,但三个种在利用的C、N源种类上有一定的差别。通过抑芽法实验和HPLC分析分别表明三种鹅膏菌所含肽类毒素在种类和含量上有所不同,但都对绿豆发芽有一定的抑制作用。A.pseudoporphyria和A.fritillaria菌丝体中α-amanitin的含量分别为35.56μg/gDCW(drycellweight细胞干重)和26.02μg/gDCW,不含有phalloidin和β-amanitin;A.muscaria菌丝体中没有检测到α-amanitin、β-amanitin和phalloidin。结果表明供试的三种鹅膏菌在基本培养条件及所产肽类毒素方面存在种水平上的差异。     

2000年以来有毒蘑菇研究新进展

误食毒蘑菇而中毒一直被认为是一个对人类健康造成威胁的全球性问题,也是我国食物中毒事件中导致死亡的最主要因素。对2000年以来在有毒蘑菇新种类、新毒素与新症状、有毒蘑菇鉴定及毒素检测新方法、有毒蘑菇中毒机理、毒素基因克隆、中毒治疗以及鹅膏肽类毒素治疗肿瘤等领域取得的新进展进行了综述,并对一些热点研究领域做了展望。     

玫瑰红鹅膏主要肽类毒素的HPLC 测定及其对白色念珠菌的抑制活性

【目的】检测玫瑰红鹅膏中所含肽类毒素及其含量,并对其肽类毒素的抑制白色念珠菌活性进行研究。【方法】采用HPLC和ESI-MS法从玫瑰红鹅膏中分离并鉴定出所含肽类毒素,并采用HPLC法测定其子实体、菌盖及菌柄和菌托混合部分中肽类毒素的含量。同时,采用纸片法研究了玫瑰红鹅膏粗毒液和分离到的单品肽类毒素对白色念珠菌JLC31680和JLC31681的抑菌作用。【结果】分离并鉴定出α-鹅膏毒肽(α-AMA)、β-鹅膏毒肽(β-AMA)和二羟鬼笔毒肽(PHD)等3种肽类毒素。玫瑰红鹅膏子实体中α-AMA、β-AMA、PHD的含量分别为30.3168、6.9932和9.9459 mg/g;菌盖中含量分别为44.9573、11.0798和11.3025 mg/g;菌柄和菌托混合部分中:α-AMA 11.6904 mg/g和PHD 7.9775 mg/g,β-AMA未检出。粗毒液、α-AMA、β-AMA和PHD对白色念珠菌JLC31680均具有很好的抑制作用,抑制率分别达到11.96%、32.52%、23.29%(p<0.01)和15.46%(p<0.05);粗毒液和β-AMA对白色念珠菌JLC31681的最高抑制率分别为10.16%和11.10%(p<0.01),α-AMA对白色念珠菌JLC31681最高抑菌率为6.89%(p<0.05)。【结论】玫瑰红鹅膏中的三种肽类毒素的含量较高,是制备肽类毒素的新资源;其具有抑制白色念珠菌的活性,可开发利用。     

Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtii: Evidence for RNA Polymerase Mutants

The inhibitory activities of amatoxins on the growth of Chlamydomonas reinhardtii have been determined using a convenient assay based upon incubation in multiwell tissue culture plates followed by turbidimetric estimates of growth on a multiwell plate reader. Values for the inhibitory dosage at which growth is 50% of untreated culture (ID₅₀) of 5.4, 6.6, and 5.6 micromolar were obtained for α-amanitin, O-methyl-α-amanitin, and amaninamide, respectively. Treatment of liquid cultures with 1 microgram per milliliter N-methyl-N′ -nitro-N-nitrosoguanidine followed by growth in agar pour tubes containing 25 micromolar α-amanitin led to the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID₅₀ values from 36 micromolar to greater than 200 micromolar. Two lines completely resistant to inhibition by 200 micromolar α-amanitin provided partially purified RNA polymerase activities that were 160-fold and 5600-fold more resistant to inhibition than the analogous enzyme activity from the wild-type strain. These studies provide evidence that Chlamydomonas reinhardtii does not contain significant activity capable of inactivating α-amanitin and that this amatoxin may be used to select for RNA polymerase mutants.     

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。     

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.     

Traceability of Amanita fuliginea poisoning using DNA barcoding and UPLC-MS/MS

Amanita fuliginea is a lethal poisonous mushroom found in Japan and southern China. The primary toxins are α-amanitin (α-AMA) and β-amanitin (β-AMA). There is a lack of systematic and comprehensive investigations on the traceability of A. fuliginea poisoning due to technological limitations. This study aimed to examine whether A. fuliginea poisoning incidents could be traced using DNA barcoding and ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS). We collected A. fuliginea specimens and prepared cooked and cooked plus simulated gastric fluid (SGF)-treated samples. We then performed DNA barcoding of internal transcribed spacer regions for species identification and UPLC-MS/MS for toxin level determination. Our results indicate that under the experimental conditions used herein, DNA barcoding can be used for molecular identification of mushroom samples that are cooked and/or cooked plus SGF-treated for less than 30 min; UPLC-MS/MS can be used for toxin analysis of cooked and SGF-treated (0–1440 min) samples. This is the first time that DNA barcoding and UPLC-MS/MS have been combined for studying the toxicological traceability of A. fuliginea using simulated gastric contents or vomit in northern China. Our data provide support for the treatment of clinical mushroom poisoning cases.     

Distribution and mechanism of α-amanitin tolerance in mycophagous Drosophila (Diptera: Drosophilidae)

Many mycophagous species of Drosophila can tolerate the mushroom poison α-amanitin in wild mushrooms and in artificial diet. We conducted feeding assays with sixteen Drosophila species and α-amanitin in artificial diet to better determine the phylogenetic distribution of this tolerance. For eight tolerant and one related susceptible species, we sequenced the gene encoding the large subunit of RNA Polymerase II, which is the target site of α-amanitin. We found no differences in the gene that could account for differences in susceptibility to the toxin. We also conducted feeding assays in which α-amanitin was combined with chemical inhibitors of cytochrome P450s or glutathione S-transferases (GSTs) in artificial diet to determine if either of these enzyme families is involved in tolerance to α-amanitin. We found that an inhibitor of GSTs did not reduce tolerance to α-amanitin, but that an inhibitor of cytochrome P450s reduced tolerance in several species. It is possible that the same cytochrome P450 activity that produces tolerance of α-amanitin might produce tolerance of other mushroom toxins as well. If so, a general detoxification mechanism based on cytochrome P450s might answer the question of how tolerance to α-amanitin arose in mycophagous Drosophila when this toxin is found in relatively few mushrooms.     

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.     

The Mechanisms Underlying α-Amanitin Resistance in Drosophila melanogaster: A Microarray Analysis

The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in D. melanogaster and perhaps in mycophagous Drosophila species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors.     

Colocalization of amanitin and a candidate toxin-processing prolyl oligopeptidase in Amanita basidiocarps

Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as α-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the "MSDIN" gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments.