Partial Characterization of a Novel Cardioinhibitory Peptide from the Brain of the Snail Helix aspersa

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin.2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure : H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH₂. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-ammo acid sequence of HCIP is identical to that of FMRFamide family peptides.3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified .     

A Novel Factor Xa-Inhibiting Peptide from Centipedes Venom

Centipedes have been used as traditional medicine for thousands of years in China. Centipede venoms consist of many biochemical peptides and proteins. Factor Xa (FXa) is a serine endopeptidase that plays the key role in blood coagulation, and has been used as a new target for anti-thrombotic drug development. A novel FXa inhibitor, a natural peptide with the sequence of Thr-Asn-Gly-Tyr-Thr (TNGYT), was isolated from the venom of Scolopendra subspinipes mutilans using a combination of size-exclusion and reverse-phase chromatography. The molecular weight of the TNGYT peptide was 554.3 Da measured by electrospray ionization mass spectrometry. The amino acid sequence of TNGYT was determined by Edman degradation. TNGYT inhibited the activity of FXa in a dose-dependent manner with an IC₅₀ value of 41.14 mg/ml. It prolonged the partial thromboplastin time and prothrombin time in both in vitro and ex vivo assays. It also significantly prolonged whole blood clotting time and bleeding time in mice. This is the first report that an FXa inhibiting peptide was isolated from centipedes venom.     

Isolation,Stabilization, and Characterization of a Toxin from Timber Rattlesnake Venom

A rapid and convenient method for the purification of a toxin from timber rattlesnake, Crotalus horridus horridus, venom using carboxymethyl cellulose ion-exchange chromatography has been devised. The toxicity of this venom component is labile, but it is stabilized by the addition of 20% V/V glycerol to the buffer solution. This toxin has a molecular weight of 15,000 + 700 as determined by SDS gel electrophoresis. It is both heat and protease resistant. Treatment of this venom component with 2-mercaptoethanol followed by G-50 Sephadex chromatography causes no loss of toxicity although incubation of the toxin with 1% SDS and 1% 2-mercaptoethanol prior to electrophoresis does result in a faster migrating species. The toxin does not affect neuromuscular junctions but does appear to act on the nervous system. It causes no local responses in mice.     

Ecological Release and Venom Evolution of a Predatory Marine Snail at Easter Island

Background

Ecological release is coupled with adaptive radiation and ecological diversification yet little is known about the molecular basis of phenotypic changes associated with this phenomenon. The venomous, predatory marine gastropod Conus miliaris has undergone ecological release and exhibits increased dietary breadth at Easter Island.

Methodology/Principal Findings

We examined the extent of genetic differentiation of two genes expressed in the venom of C. miliaris among samples from Easter Island, American Samoa and Guam. The population from Easter Island exhibits unique frequencies of alleles that encode distinct peptides at both loci. Levels of divergence at these loci exceed observed levels of divergence observed at a mitochondrial gene region at Easter Island.

Conclusions/Significance

Patterns of genetic variation at two genes expressed in the venom of this C. miliaris suggest that selection has operated at these genes and contributed to the divergence of venom composition at Easter Island. These results show that ecological release is associated with strong selection pressures that promote the evolution of new phenotypes.     

Design and In Silico Evaluation of a Novel Cyclic Disulfide-Rich anti-VEGF Peptide as a Potential Antiangiogenic Drug

International Journal of Peptide Research and Therapeutics - The vascular endothelial growth factor (VEGF) signaling pathway has a crucial role in regulating tumor angiogenesis. VEGF-A shows...     

Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus

Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs’ mechanism and application.     

Identification and Structural Characterization of a New Three-Finger Toxin Hemachatoxin from Hemachatus haemachatus Venom

Snake venoms are rich sources of biologically active proteins and polypeptides. Three-finger toxins are non-enzymatic proteins present in elapid (cobras, kraits, mambas and sea snakes) and colubrid venoms. These proteins contain four conserved disulfide bonds in the core to maintain the three-finger folds. Although all three-finger toxins have similar fold, their biological activities are different. A new three-finger toxin (hemachatoxin) was isolated from Hemachatus haemachatus (Ringhals cobra) venom. Its amino acid sequence was elucidated, and crystal structure was determined at 2.43 Å resolution. The overall fold is similar to other three-finger toxins. The structure and sequence analysis revealed that the fold is maintained by four highly conserved disulfide bonds. It exhibited highest similarity to particularly P-type cardiotoxins that are known to associate and perturb the membrane surface with their lipid binding sites. Also, the increased B value of hemachotoxin loop II suggests that loop II is flexible and may remain flexible until its interaction with membrane phospholipids. Based on the analysis, we predict hemachatoxin to be cardiotoxic/cytotoxic and our future experiments will be directed to characterize the activity of hemachatoxin.     

Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin

Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, secretes several virulence factors, among which is the adenylate cyclase toxin (CyaA) that plays a crucial role in the early stages of human respiratory tract colonization. CyaA invades target cells by translocating its catalytic domain directly across the plasma membrane and overproduces cAMP, leading to cell death. The molecular process leading to the translocation of the catalytic domain remains largely unknown. We have previously shown that the catalytic domain per se, AC384, encompassing residues 1–384 of CyaA, did not interact with lipid bilayer, whereas a longer polypeptide, AC489, spanning residues 1–489, binds to membranes and permeabilizes vesicles. Moreover, deletion of residues 375–485 within CyaA abrogated the translocation of the catalytic domain into target cells. Here, we further identified within this region a peptidic segment that exhibits membrane interaction properties. A synthetic peptide, P454, corresponding to this sequence (residues 454–485 of CyaA) was characterized by various biophysical approaches. We found that P454 (i) binds to membranes containing anionic lipids, (ii) adopts an α-helical structure oriented in plane with respect to the lipid bilayer, and (iii) permeabilizes vesicles. We propose that the region encompassing the helix 454–485 of CyaA may insert into target cell membrane and induce a local destabilization of the lipid bilayer, thus favoring the translocation of the catalytic domain across the plasma membrane.     

A Novel Neurotoxin from Venom of the Spider,Brachypelma albopilosum

Spiders have evolved highly selective toxins for insects. There are many insecticidal neurotoxins in spider venoms. Although a large amount of work has been done to focus on neurotoxicity of spider components, little information, which is related with effects of spider toxins on tumor cell proliferation and cytotoxicity, is available for Brachypelma albopilosum venom. In this work, a novel spider neurotoxin (brachyin) was identified and characterized from venoms of the spider, Brachypelma albopilosum. Brachyin is composed of 41 amino acid residues with the sequence of CLGENVPCDKDRPNCCSRYECLEPTGYGWWYASYYCYKKRS. There are six cysteines in this sequence, which form three disulfided bridges. The serine residue at the C-terminus is amidated. Brachyin showed strong lethal effects on American cockroaches (Periplaneta americana) and Tenebrio molitor (common mealbeetle). This neurotoxin also showed significant analgesic effects in mice models including abdominal writhing induced by acetic acid and formalin-induced paw licking tests. It was interesting that brachyin exerted marked inhibition on tumor cell proliferation.     

蕲蛇蛇毒中一个新的类凝血酶的分离纯化与表征

使用DEAE－Sephadex A-50、POROS 50HS及POROS 20PI等色谱分离技术,从蕲蛇粗毒中分离提纯得到1个具有凝血活力的组分,经SDS－PAGE和PAGE检测均为一条带,非还原条件下相对分子质量24.3kDa,还原条件下相对分子质量33.0kDa;其凝血活力为91.0NIH u/mg。该组分不具有激活因子ⅩⅢ的活性,肝素不影响该组分的凝血活性,EDTA部分抑制其活性,苯甲基磺酰氟（PMSF）则会产生不可逆抑制作用,该酶N－末端氨基酸序列为VIGGNGXDINEHRFLVAFF,经分析表明该酶是一种新的类凝血酶。     

尖吻蝮蛇蛇毒出血毒素的纯化与部分性质

目的　被尖吻蝮蛇 （ Ｄｉｅｎａｇｋｉｓｔｒｏｄｏｎ ａｃｕｔｕｓ） 咬伤会引起严重的出血, 对蛇毒出血毒素的研究有利于治疗蛇伤出血药物筛选。 方法　采用 Ｓｅｐｈａｄｅｘ Ｇ７５, Ｄ Ｅ Ａ Ｅ Ｓｅｐｈａｄｅｘ Ａ５０, Ｓｅｐｈａｄｅｘ Ｇ２００ 和两次 Ｐ Ｂ Ｅ 聚焦层析纯化。 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳和等电聚焦电泳测定纯化样品的纯度和等电点。氨基酸组成用自动氨基酸分析仪测定。以小鼠背部皮下注射部位出血斑的面积来确定最小出血剂量和常规的方法测定酶活性。结果　从尖吻蝮蛇毒中纯化到一个相对分子量为５６ ０００ 的出血毒素 （ Ｄａ Ｈ Ｔ３）, 经氨基酸组成测定计算,它由 ４８７ 个氨基酸残基组成。此成分在 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ上显示出一条均一的蛋白染色带, 其ｐ Ｉ为５５０。该出血成分的最小出血剂量是 ２６μｇ, 具有蛋白水解酶活力, 其活力为 ３６８, 但没有精氨酯酶和磷脂酶 Ａ２ 活力。当加入 Ｅ Ｄ Ｔ Ａ 螯合剂去除金属离子后, 它们的出血活力和蛋白水解酶活力均丧失。 结论　这是从大陆尖吻蝮蛇毒中获得的一个新的出血金属蛋白酶 （ Ｄａ Ｈ Ｔ３）。     

竹叶青蛇毒L-氨基酸氧化酶的纯化、诱导细胞凋亡和抗菌作用

经过分子筛层析和离子交换层析,我们从竹叶青(Trimeresurus stejnegeri)蛇毒中纯化得到了竹叶青蛇毒L-氨基酸氧化酶(TSV-LAO)。实验表明,TSV-LAO是一种糖蛋白,其分子由非共价连接的两个相同的亚基组成,SDS-聚丙烯酰氨凝胶电泳一个亚基的表观分子量为58 kDa。TSV-LAO酶比活力为1100 U/mg,其分子脱掉糖基化成分后不影响酶活力。TSV-LAO在浓度为1.0μg/mL以上时可以诱导C8166细胞凋亡。TSV-LAO对实验病原微生物具有选择性抗生作用并具有明显的量效关系,对白色念珠菌(ATCC2002)、金黄色葡萄球菌(ATCC2592)和短小芽孢杆菌(CMCCB11207)的最小抑菌浓度分别是0.3,0.4和1.0μg/mL,即使在最高实验浓度10μg/mL,TSV-LAO对其它实验菌株也未显示抑菌作用。     

尖吻蝮蛇蛇毒出血毒素DaHT-3的纯化与性质

被尖吻蝮蛇咬伤会引起严重的出血,对蛇毒出血毒素的研究有利于治疗蛇伤出血药物的筛选.通过SephadexG-75,DEAE-SephadexA-50,SephadexG-200和两次PBE聚焦层析,从尖吻蝮蛇(Dienagkistrodonacutus)蛇毒中纯化到一个分子量为56000的出血毒素(DaHT-3),经氨基酸组成测定计算,由487个氨基酸残基组成.此成分在SDS-PAGE上显示出一条均一的蛋白染色带,用等电聚焦电泳测定,其pI为5.50.该出血成分的最小出血剂量是2.6μg,具有蛋白水解酶活力,其活力为3.68,但没有精氨酸酯酶和磷脂酶A2活力.用红外光谱仪研究DaHT-3在溶液中酰胺I带的吸收谱,该毒素含有31.8%的α螺旋、56.1%的β折叠和12.1%的转角;当加入EDTA螯合剂去除金属离子后,它们的α螺旋、β折叠、转角和无规卷曲分别变为11%、26.4%、46.2%和16.5%,而出血活力和蛋白水解酶活力均丧失,表明该出血毒素是金属蛋白酶     

蕲蛇蛇毒中一种新型金属蛋白酶AA-MP-I的纯化和表征

本研究通过嗜硫色谱、Sephadex G-75、蓝胶和POROS HQ20离子交换色谱,从蕲蛇蛇毒中分离得到一种新组分AA-MP-I。该酶为分子量22.9kDa的单体蛋白,等电点为5.55,不含中性糖基,N端序列为STE-FQRYMEIVIVVDHSMVK,结果表明其为新型P-I型金属蛋白酶,对温度敏感,具有抗凝血活性,40℃下抗凝血活性最强,具有出血毒性,无磷脂酶A2活性。     

Identification and Characterization of a Novel Staphylococcal Emetic Toxin

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.     

Purification and Characterization of Phosphoribulokinase from the Marine Chromophytic Alga Heterosigma carterae

In this study we characterized phosphoribulokinase (PRK, EC 2.7.1.19) from the eukaryotic marine chromophyte Heterosigma carterae. Serial column chromatography resulted in approximately 300-fold purification of the enzyme. A polypeptide of 53 kD was identified as PRK by sequencing the amino terminus of the protein. This protein represents one of the largest composite monomers identified to date for any PRK. The native holoenzyme demonstrated by flow performance liquid chromatography a molecular mass of 214 ± 12.6 kD, suggesting a tetrameric structure for this catalyst. Because H. carterae PRK activity was insensitive to NADH but was stimulated by dithiothreitol, it appears that the enzyme may require a thioredoxin/ferredoxin rather than a metabolite mode of regulation. Kinetic analysis of this enzyme demonstrated Michaelis constant values of ribulose-5-phosphate (226 μm) and ATP (208 μm), respectively. In summary, H. carterae PRK is unique with respect to holoenzyme structure and function, and thus may represent an alternative evolutionary pathway in Calvin-cycle kinase development.     

Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

Background

It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined.

Methodology/Principal Findings

We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0–10.0 and at temperatures ≤40°C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C₂–C₈), and its suitable substrate was p-nitrophenyl caproate (C₆) with optimal catalytic conditions of 39°C and pH 8.0.

Conclusions/Significance

Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis.     

Characterization of a Novel Putative S-Adenosylmethionine Decarboxylase-Like Protein from Leishmania donovani

In addition to the S-adenosylmethionine decarboxylase (AD) present in all organisms, trypanosomatids including Leishmania spp. possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL). Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In Trypanosoma ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from Leishmania spp. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of Leishmania donovani (L. donovani) ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, L. donovani ADL binds S-adenosylmethionine (SAM) and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. In silico protein-protein interaction study reveals that L. donovani ADL can interact with AD. These results indicate that L. donovani ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.     

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a K_m value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.Many pathogenic bacteria and fungi produce toxins that interfere with various functions of plant cells and may affect plant defense mechanisms (Durbin, 1981). Toxin production is commonly associated with disease severity and can be involved in colonization or systemic invasion by the pathogen (Schäfer, 1994). Toxin resistance has been shown in most cases to be based on the ability of the plant to metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance genes that encode proteins involved in detoxification mechanisms have been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and Birch, 1997). In many cases a relationship exists between toxin tolerance and resistance to the disease (Anzai et al., 1989; Meeley et al., 1992). The availability of toxin-resistance genes will permit a greater understanding of the mechanisms causing plant disease and will also set the stage for engineering resistance to plant disease (Keen, 1993).Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by the ascomycete fungus Eutypa lata (Pers.: Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991). This disease is responsible for considerable loss in yield and is the most devastating disease of grapevine (Vitis vinifera) in many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The fungus infects the stock through pruning wounds and is present in the xylem and phloem of the vine trunk and branches (Moller and Kasamitis, 1978; Duthie et al., 1991). After a long incubation period, a canker forms around the infected wound. The toxin synthesized by the fungus in the trunk is believed to be transported by the sap to the herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates grapevine cells through passive diffusion and its accumulation in the cytoplasm has been explained by an ion-trapping mechanism related to the ionization state of the molecule (Deswarte et al., 1996b). In the cell the effects of eutypine include reduction of adenylated nucleotide content, inhibition of succinate dehydrogenase, uncoupling of oxidative phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to dwarfed and withered new growth of branches, marginal necrosis of the leaves, dryness of the inflorescence, and, finally, death of one or more branches (Moller and Kasamitis, 1981). The toxin appears to be an important virulence factor involved in symptom development of the disease (Deswarte et al., 1996a). However, the absence of toxin-deficient mutants of the fungus and its long incubation period in the trunk before symptom development have prevented a critical study of the toxin in vine plants. Determining the gene responsible for eutypine resistance would therefore be an important critical tool in determining the role of eutypine toxin in symptom development in the disease; and it has the potential to confer resistance to transgenic grapevines.Recently, Colrat et al. (1998) found detoxification to occur in grapevine cells through the enzymatic reduction of eutypine into its corresponding alcohol, eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol). We have determined that this derivative of the toxin is nontoxic for grapevine tissues. Furthermore, we have established a relationship between the susceptibility of grapevine to eutypa dieback and the ability of tissues to inactivate eutypine, suggesting that the detoxification mechanism plays an important role in defense reactions. Eutypine is enzymatically detoxified in numerous plant species and, among them, we found that the tissues of mung bean (Vigna radiata), a nonhost plant for the pathogen, exhibit an efficient detoxification activity. As a prerequisite for demonstrating the involvement of eutypine toxin in eutypa dieback, we report here the purification to homogeneity and the characterization of an ERE from etiolated mung bean hypocotyls.