Biological and Chemical Detection of Trichothecene Mycotoxins of Fusarium Species

The procedure for biological and chemical detection of trichothecene-type mycotoxins and its application to the screening of Fusarium for toxic strains were described.     

Production of Trichothecene Mycotoxins by Fusarium Species in Shake Culture

Twelve T-2 toxin-producing isolates and four fusarenon-X-producing isolates of Fusarium species were examined for their ability to produce trichothecene mycotoxins in shake culture and jar fermentation. T-2 toxin producers such as Fusarium solani, F. sporotrichiodes, and F. tricinctum produced T-2 toxin and neosolaniol in semisynthetic medium. F. solani M-1-1 produced the largest amount of the mycotoxins in a nutrient medium consisting of 5% glucose (or sucrose), 0.1% peptone, and 0.1% yeast extract in either shake culture or jar fermentation at 24 to 27 C for 5 days. None of the isolates produced significant amount of fusarenon-X in shake cultures.     

Detection of Airborne Stachybotrys chartarum Macrocyclic Trichothecene Mycotoxins on Particulates Smaller than Conidia

Highly respirable particles (diameter, <1 μm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborne Stachybotrys chartarum trichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluent Stachybotrys growth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P < 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P < 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show that S. chartarum trichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.     

Detection of Airborne Stachybotrys chartarum Macrocyclic Trichothecene Mycotoxins in the Indoor Environment

The existence of airborne mycotoxins in mold-contaminated buildings has long been hypothesized to be a potential occupant health risk. However, little work has been done to demonstrate the presence of these compounds in such environments. The presence of airborne macrocyclic trichothecene mycotoxins in indoor environments with known Stachybotrys chartarum contamination was therefore investigated. In seven buildings, air was collected using a high-volume liquid impaction bioaerosol sampler (SpinCon PAS 450-10) under static or disturbed conditions. An additional building was sampled using an Andersen GPS-1 PUF sampler modified to separate and collect particulates smaller than conidia. Four control buildings (i.e., no detectable S. chartarum growth or history of water damage) and outdoor air were also tested. Samples were analyzed using a macrocyclic trichothecene-specific enzyme-linked immunosorbent assay (ELISA). ELISA specificity was tested using phosphate-buffered saline extracts of the fungal genera Aspergillus, Chaetomium, Cladosporium, Fusarium, Memnoniella, Penicillium, Rhizopus, and Trichoderma, five Stachybotrys strains, and the indoor air allergens Can f 1, Der p 1, and Fel d 1. For test buildings, the results showed that detectable toxin concentrations increased with the sampling time and short periods of air disturbance. Trichothecene values ranged from <10 to >1,300 pg/m³ of sampled air. The control environments demonstrated statistically significantly (P < 0.001) lower levels of airborne trichothecenes. ELISA specificity experiments demonstrated a high specificity for the trichothecene-producing strain of S. chartarum. Our data indicate that airborne macrocyclic trichothecenes can exist in Stachybotrys-contaminated buildings, and this should be taken into consideration in future indoor air quality investigations.     

Biological Modification of Trichothecene Mycotoxins: Acetylation and Deacetylation of Deoxynivalenols by Fusarium spp

Attempts were made to elucidate the acetyl transformation of novel trichothecene mycotoxins, 3a,7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one (deoxynivalenol) and its derivatives, by trichothecene-producing strains of Fusarium nivale, F. roseum, and F. solani. In the peptone-supplemented Czapek-Dox medium, F. roseum converted 3a-acetoxy-7a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (3-acetyldeoxynivalenol) to deoxynivalenol. 3-Acetyldeoxynivalenol was also deacetylated by intact mycelia of the three strains in sugar-free Czapek-Dox medium. The growing F. nivale acetylated deoxynivalenol to afford a small amount of 3-acetyldeoxynivalenol. 3a,7a,15-Triacetoxy-12,13-epoxytrichothec-9-en-8-one (7,15-diacetyl-deoxynivalenol), which was then deacetylated to give 7a-acetoxy-3a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (7-acetyldeoxynivalenol). It was noted that the ester at C-7 was not hydrolyzed by the fungal mycelium.     

农产品中主要真菌毒素微生物及微生物酶解毒研究与发展趋势

真菌毒素是一类丝状真菌次级代谢产物，为高毒性天然污染物，在农产品生产和贮运过程中难以完全消除，污染率高、危害性大，已成为农产品安全主要风险因子之一。农产品中主要真菌毒素生物解毒研究对污染原料再利用、动物健康养殖和食品安全具有重要意义。研究表明，微生物菌株或微生物酶对真菌毒素可进行高效地降解转化或生物吸附。近十年，主要真菌毒素生物解毒研究已逐渐成为真菌毒素污染控制领域的关注重点，但关于生物降解机制的研究仍处于初步发展阶段，很多微生物菌种的降解转化机制仍不清楚。本文将从农产品中主要真菌毒素生物降解菌种、生物解毒酶及作用方式等方面进行总结，以期为生物解毒深入研究及产业化技术发展提供思路。     

Effect of Chemical Chaperones on Glucose-Induced Lysozyme Modifications

Nonenzymatic glycation of biomacromolecules occurs due to the diabetes mellitus and ageing. A number of small molecules, known as chemical chaperones, stabilize protein conformation against thermal and chemically induced denaturation. These compounds are including: polyamines (e.g. spermine and spermidine), amino acids (e.g. lysine) and polyols (e.g. glycerol). In this study the effect of spermidine (Spd), spermine (Spm), and glycerol on glycation, structure and function of lysozyme (LZ), as an extra-cellular protein, by different techniques is investigated. LZ is incubated with or without glucose (50 or 100 mM) in the absence or presence of Spd/Spm/glycerol at 37 °C up to 16 weeks. All the observed changes of glycated-LZ in comparison with the native protein, including: increased fluorescence emission, alteration in the secondary and tertiary structure, and reduced electrophoretic mobility- indicate its structural changes that are accompanied with its reduced activity. Glucose in the presence or absence of Spd induces the protein dimerization, but glucose plus Spm induces its trimmerization. In contrast, glycerol inhibits the LZ glycation and prevents the large changes on its structure and function. Glucose binds lysine residues, decreases the protein positive charges and induces some alterations in its structure and activity. Polyamines also directly bind to LZ, increase its positive charges and hence induce more glycation; more conformational changes, oligomerization and its inactivation in the presence of glucose, but glycerol affect the protein environment and preserve protein from these harmful effects.     

Antiviral Activity of Trichothecene Mycotoxins (Deoxynivalenol,Fusarenon-X,and Nivalenol) against Herpes Simplex Virus Types 1 and 2

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC₅₀) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.     

Perspectives for Chemical Modifications of Enzymes

Abstract

The contents of this review include current information on advances regarding the chemistry and chemical modifications (surface and internal) of enzymes that affect their kinetic behavior. As an illustrative example of identifying catalytically essential amino acid residues at the active site(s), the case of liver alcohol dehydrogenase is shown.     

蛋白酶体翻译后修饰功能机制研究

蛋白酶体是真核细胞内介导蛋白质特异性降解的主要复合物,在蛋白质质量控制和细胞稳态维持中发挥关键作用.研究发现,蛋白酶体含量或功能的异常会导致癌症、神经退行性疾病等诸多人类恶性疾病.围绕蛋白酶体的活性调控,已经发展了多种靶向药物,加强对蛋白酶体活性精确调控机制的研究具有重要的学术价值与临床意义.蛋白酶体的含量、组装及其活...     

微生物混合发酵的研究及应用

由于不同微生物之间的正相互作用,人们发现应用两种或两种以上微生物混合发酵能更好地解决实践中的许多问题。在过去几年中,对微生物的混合发酵的应用以及其中微生物之间的相互作用机理的研究取得了明显进展,主要有以下4个方面:(1)对生物质的降解利用;(2)对环境污染物的降解;(3)生产特定的代谢产物;(4)混合发酵的工艺。综述了微生物混合发酵的应用及相关机理、涉及的微生物和影响因素。     

胡杨、灰叶胡杨花粉成分及生活力的比较研究

比较研究了胡杨,灰叶胡杨花粉的化学成分及2种花粉在不同贮藏条件下的生活力变化。结果表明;在室温,4℃冰箱内保存,灰叶胡杨花粉生活力保持的时间均比胡杨花粉生活力保持的时间长,同一个种的花粉贮藏相同时间,在4℃冰箱内贮藏的生活力比室温下贮藏的生活力强,耐贮藏的灰叶胡杨花粉水分,淀粉含量较胡杨花粉高,蛋白质,还原糖,17种游离氨基酸含量较胡杨花粉含量低。     

A Simple Method with Liquid Chromatography/Tandem Mass Spectrometry for the Determination of the Six Trichothecene Mycotoxins in Rice Medium

A selective and speedy LC–MS/MS method was developed to determine six trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin) in rice medium where Fusarium graminearum were cultivated for in vitro tests. The analytes were extracted from the rice medium with acetonitrile/water (85/15, v/v), and diluted with acetonitrile/water (5/95, v/v) in order to minimize the effects of matrices. Diluted solutions were analyzed by LC–MS/MS with electrospray ionization (ESI) interface in negative or positive ion mode and the multiple reaction monitoring mode. Recovery rates were 76–106% with a spiked level at 1–6 μg/kg of mycotoxins that corresponded to the limit of quantitation. The method was applied to study the time courses of trichothecene production and the biomass of fungi by three Fusarium graminearum strains. Three strains have different mycotoxin biosynthesis pathways, wFg14 and 03E-1 were DON producer, and 03N-1 was NIV producer.     

Chemical Modifications of Melatonin Receptors in Chicken Brain

Abstract: The membrane-bound or solubilized melatonin receptors were treated with protein-modifying agents under specific conditions and then assayed for ¹²⁵I-melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl ( N -ethylmaleimide and p -chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1-fluoro-2,4-dinitrobenzene) inhibited ¹²⁵I-melatonin binding in a dose-dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of ¹²⁵I-melatonin binding to guanine nucleotides after N -ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N -acetylimidazole, and p -nitrobenzenesulfonyl fluoride also inhibited ¹²⁵I-melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross-reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited ¹²⁵I-melatonin binding in a dose-dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.     

Comparative Studies on the Sampling and Investigation of Microbial Contamination of Surfaces by the Contact Plate and Swab Methods

In this study a comparison was made of recovering bacteria from stainless steel, plastic, wood, agar and meat surfaces. Sampling was performed using the agar contact and swab methods. The results indicated that for a flat, firm surface the contact plate method was more suitable, considering both recovery and repeatability. Swabbing was, by contrast, better for flexible and uneven surfaces and for heavily contaminated surfaces. Bacteriological guidelines are suggested for the hygienic evaluation of surface contamination of meat carcasses and for working surfaces in meat processing plates.     

Studies on Antioxidative Substances of Microbial Origin

Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80°C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus.     

京尼平苷的微生物转化研究

本文利用高产β-葡萄糖苷酶菌种制备游离细胞和固定化细胞,在温和条件下可将京尼平苷转化为京尼平,转化率高达98%.通过条件优化,得到了最优转化条件:京尼平添加量1.5%,pH自然,摇瓶装量20%,28 ℃、150 rpm转化24 h.固定化细胞可连续使用4次.这种微生物转化法安全、高效,产品纯度高,是生产京尼平的一种新方法.     

Studies on Microbial Metabolisms of Sugar Nucleotides

Effects of various factors including incubation time, water content of airdried cells, concentration and pH of KH₂PO₄–K₂HPO₄ mixture, d-glucose concentration, MgSO₄ concentration, GMP concentration, cell concentration, aeration and various kinds of carbohydrates on the fermentative production of GDP-mannose, GDP and GTP from 5′-GMP by air-dried cells of baker’s yeast were investigated. The water content of air-dried cells was the most important factor in the fermentation. When the air-dried cells of baker’s yeast (100 mg/ml) were incubated with 5′-GMP (20 μmoles/ml), d-glucose (800 μmoles/ml), potassium phosphate buffer (360 μmoles/ml, pH 7.0), and MgSO₄ (20 μmoles/ml), 2-hr incubation gave GDP in 20% yield and GTP in 61.1% yield, GDP-mannose being produced in 45% yield after 8-hr incubation. The phosphorylation of 5′-AMP, 5′-dAMP, 5′-dGMP 5′-CMP and 5′-UMP was also observed in high yields under the same conditions.     

Studies on Microbial Metabolism of Sugar Nucleotides

The fermentative production of uridine diphosphate N-acetylglucosamine (UDPAG) from 5′-UMP and glucosamine by dried cells of baker’s yeast was studied. UDPAG was found to accumulate in a reaction system containing 5′-UMP, glucosamine, fructose, inorganic phosphate and magnesium ions with dried baker’s yeast as an enzyme source. UDPAG was separated from the reaction mixture by means of anion exchange column chromatography and was identified by several biochemical methods.

The reaction conditions for the fermentative production of UDPAG were examined. The yield of UDPAG was about 40~66% based on 5′-UMP added when fermentation conditions were optimized. The concentration of glucosamine and potassium phosphate buffer, and pH as well as the water content of dried cells greatly affected the formation of UDPAG.