A Model Transgenic Cereal Plant with Detoxification Activity for the Estrogenic Mycotoxin Zearalenone

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the necrotrophic cereal pathogen Fusarium graminearum. This mycotoxin is detoxified by ZHD101, a lactonohydrolase from Clonostachys rosea, or EGFP:ZHD101, its fusion to the C-terminus of an enhanced green fluorescence protein. We previously showed that egfp:zhd101 is efficiently expressed in T₀ leaves of rice. In this study, we assessed the feasibility of in planta detoxification of the mycotoxin using progeny. When protein extract from T₁ leaves was incubated with ZEN, the amount of the toxin decreased significantly as measured by HPLC. ZEN degradation activity was also detected in vivo in transgenic T₂ seeds. These results suggest that zhd101 can be exploited as an efficient and cost-effective system for protection of important cereals that are more susceptible to the pathogen (e.g., wheat and maize) from contamination with the estrogenic mycotoxin.     

高通量筛选雌激素类化合物重组绿色荧光蛋白酵母细胞测评体系

以载体双表达的方式构建重组酵母环境雌激素的评价体系, 用于快速筛选雌激素类化合物。在表达载体中, 用3-磷酸甘油醛脱氢酶(GPD)启动子驱动a人雌激素受体基因(hERa)的表达; 在报告载体中, 用雌激素效应元件(ERE)调控的绿色荧光蛋白(yEGFP)作为报告基因。将两者转化于酵母细胞(W303-1A)中, 构建成重组绿色荧光蛋白酵母细胞。该酵母细胞经不同浓度的雌激素类化合物作用后, 发现GFP的表达量与此类受试物具有明显的剂量效应关系。与其他环境雌激素酵母评价体系相比, 该重组酵母评价细胞, 在应用时不需要破坏细胞壁, 也不需要底物和相关试剂, 可直接在96孔板中操作完成, 具有快速、高通量、敏感性高、重现性好及廉价等特点。     

An Inexpensive Apparatus Facilitating the Storage of Oxygen Sensitive Compounds

The design, construction, and use of an inexpensive apparatus which facilitates the storage of oxygen sensitive compounds in serum bottles is described.     

A Yeast Bioassay for Androgenic and Anti-Androgenic Compounds Based on the NH2- and COOH-Terminal Interaction of Androgen Receptor

Androgenic compounds induce an interaction between the NH₂- and COOH-terminal regions (N–C interaction) of androgen receptor (AR). We describe a rapid yeast bioassay for androgenic and anti-androgenic compounds based on androgen-dependent β-catenin-enhanced N–C interaction. The bioassay was also effective at detecting compounds that inhibit the N–C interaction in ways that do not involve binding to the ligand-binding domain.     

Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17β-estradiol over the concentration range of 1.2 × 10⁻⁸ through 5.6 × 10⁻¹² M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) × 10⁻¹⁰ and (2.4 ± 1.0) × 10⁻¹⁰ M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10⁻¹¹ to 2.8 × 10⁻⁹ M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.     

利用GFP表达系统检测雌激素类化合物的研究

根据雌激素类化合物的转录调节原理,构建受雌激素应答元件（ERE）调控的3×ERE EGFP-N1重组报告基因载体,转染雌激素受体阳性的MCF-7乳腺癌细胞,分离出稳定转染的细胞克隆ERE-GFP-MCF7,将不同浓度的雌二醇（E2）及其他化合物加入稳定转染的细胞后检测GFP表达水平的变化。雌二醇（E2）以剂量依赖的方式诱导稳定转染细胞ERE-GFP-MCF7表达GFP,最大效应浓度为1×10－10mol/L ,EC50为1.5×10－11 mol/L;已知的植物雌激素大豆甙元和白藜芦醇同样以剂量依赖的方式诱导GFP的表达,EC50分别为2.4×10－7 mol/L和6.2×10－6 mol/L,而葡萄籽多酚没有明显的雌激素样活性。雌激素拮抗剂他莫西芬以剂量依赖的方式抑制雌二醇诱导GFP的表达,最大抑制浓度为1×10－7 mol/L。利用此细胞模型检测不同化合物诱导的GFP表达强度可快速检测雌激素受体的配体。     

Development and Application of an Immunoaffinity Column Enzyme Immunoassay for Mycotoxin Zearalenone in Complicated Samples

The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC₅₀ against ZEA of 0.02 µg L⁻¹. The mAb 2D3 exhibited a high recognition of ZEA (100%) and β-zearalenol (β-ZOL, 88.2%). Its cross-reactivity with α-zearalenol (α-ZOL) and β-zearalanol (β-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83–93%) and HPLC (94–108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize.     

A Sensitive Bioassay for the Detection of Cry1A Toxin Expression in Transgenic Cotton

In the process of development of insect resistant transgenic plants and also to evaluate the consistency in expression of the toxin under greenhouse and field conditions, immunological and bioassays are commonly used. The assay being described in this report, is based on the high levels of sensitivity of a cotton leaf feeding insect, the semilooper, Anomis flava (Fabricius) to Cry toxins (Cry1Aa, Cry1Ab and Cry1Ac). The assay is sensitive, quick and reproducible. Cry1Ac was the most toxic followed by Cry1Ab and Cry1Aa. LC 50 s of the three toxins on first instar larvae ranged from 0.79-6.08 ng cm -2 of leaf. LC 50 s of Cry1Ac for the fourth instar larvae ranged from 12.91-21.14 ng cm -2 while LC 50 s for Cry1Aa and Cry1Ab were in the range 53.0-138 ng cm -2 . The fiducial limits (at 95% probability) of the probit assay data indicated that there was no difference in response between the three different populations to each of the three toxins. The data from all assays were pooled for each of the three toxins separately and subjected to regression analysis to obtain a cumulative log dose response for first and fourth instar larvae. These can be used as standard curves to quantify toxin expression in plants based on mortality response of either first or fourth instar A. flava larvae. Apart from being used to detect expression in putative Bt cotton transgenic plants, the assay can also be used to follow the activity of Cry toxins in transgenic cotton plants in the field during the growing season.     

A New Sensitive Bioassay for Determination of Microbially Available Phosphorus in Water

The content of assimilable organic carbon has been proposed to control the growth of microbes in drinking water. However, recent results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking waters. Even a very low concentration of phosphorus (below 1 μg of P liter⁻¹) can promote extensive microbial growth. We present here a new sensitive method to determine microbially available phosphorus concentrations in water down to 0.08 μg of P liter⁻¹. The method is a bioassay in which the analysis of phosphorus in a water sample is based on maximum growth of Pseudomonas fluorescens P17 when the energy supply and inorganic nutrients, with the exception of phosphorus, do not limit bacterial growth. Maximum growth (CFU) in the water sample is related to the concentration of phosphorus with the factor 373,200 ± 9,400 CFU/μg of PO₄-P. A linear relationship was found between cell growth and phosphorus concentration between 0.05 to 10 μg of PO₄-P liter⁻¹. The content of microbially available phosphorus in Finnish drinking waters varied from 0.1 to 10.2 μg of P liter⁻¹ (median, 0.60 μg of P liter⁻¹).     

Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (a_w) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an a_w of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.     

A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen⁻¹. This amount corresponds to 10⁴ to 10⁵ CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10⁸ CFU ml⁻¹. The detection limit for food was 3 g of rice containing 10⁶ to 10⁷ CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen⁻¹, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.     

Bioassay for Hamycin and Amphotericin B in Serum and Other Biological Fluids

A bioassay suitable for measuring concentrations of the polyene antifungal agents hamycin and amphotericin B in biological fluids is described. By using Paecilomyces varioti as the indicator organism, sensitivity of the bioassay was found to be in the range of 0.01 to 0.02 mug/ml. A linear dose-response curve was obtained with amphotericin B; the curve for hamycin was curvilinear. In a series of assays, hamycin serum levels in the range of 0.01 to 3.5 mug/ml were measured; with amphotericin B, serum levels in the range of 0.015 to 0.175 mug/ml were measured in patients receiving orthodox intravenous medication and as high as 9.0 mug/ml in one patient treated with extraordinarily high doses of the drug.     

The Use of Baker's Yeast in the Generation of Asymmetric Centers to Produce Chiral Drugs and Other Compounds

ABSTRACT:?

This review gives a general idea about the importance of chiral carbon in medicine and a way to obtain chiral building blocks with Baker's yeast (Saccharomyces cerevisiae) or synthesis of medicaments and other organic compounds. Reactions with these microorganisms are cheaper and easier to be executed than with chemicals (e.g., organometallics). Examples of important and practical reactions catalyzed by enzymes inside Saccharomyces cerevisiae are given and probable mechanisms of action of these enzymes are shown. Although these microbes have advantages such as low cost and availability, there are some concerns that are necessary to be resolved, such as NAD(P)H dosage to choose strains more adequate for reduction reactions.     

A Review of an Ultrafast and Sensitive Bioassay Platform Technology: Microwave-accelerated Metal-enhanced Fluorescence

Since the publication of our first paper on the microwave-accelerated metal-enhanced fluorescence (MAMEF) bioassay platform technology in 2005 (Aslan and Geddes, Anal Chem 77:8057–8067, 2005), we have been repeatedly asked to comment on the advantages of “microwave heating” with plasmonic nanostructures over conventional heating for bioassays by many of our colleagues in the community. We note that one can find a couple of review articles, one by Mingos (Gabriel et al., Chem Soc Rev 27:213–223, 1998) and another by Thostenson and Chou (Manufacturing 30:1055–1071, 1999), summarizing the fundamentals and several applications of microwave processing of chemical compounds and composite materials, respectively. These review articles also present a direct comparison of microwave heating with conventional heating with respect to the processing of materials and microwave-assisted synthesis of chemical compounds. In this review article, we seek to remind the reader of the fundamentals of microwave heating and the interactions of microwaves with chemical and biological materials relevant to our recent work on bioassays, rather than repeating the information provided in the above-mentioned very informative reviews. We also summarize our work on MAMEF-based bioassays where we use plasmonic nanostructures to additionally plasmon-enhance fluorescence signatures.     

A New Rapid and Sensitive Bioluminescence Assay for Monoamine Oxidase Activity

The in vivo luminescence of an aldehyde-requiring mutant of the luminous bacteria Vibrio harveyi (M42) increases dramatically upon the addition of long-chain aliphatic aldehydes (C8-C16). The intensity of this luminescence is linearly related to aldehyde concentration. This property was utilized for the determination of monoamine oxidase activity using n-octylamine and n-decylamine as substrates, which are converted by monoamine oxidase to n-octylaldehyde and n-decylaldehyde, respectively. The addition of the amine to a suspension containing rat liver mitochondria and M42 cells initiated a luminescence that was directly proportional to monoamine oxidase activity according to two parameters: (1) the rate of the initial increase in luminescence and (2) the final "steady-state" level of luminescence. The new assay has advantages of high sensitivity, rapidity, the possibility to perform discontinuous as well as continuous monitoring of monoamine oxidase activity, and applicability to turbid preparations.     

A Rapid,Sensitive, Seedling-Based Bioassay for the Determination of Toxicity of Solid and Liquid Substrates and Plant Tolerance

We have developed a rapid, sensitive, seedling-based bioassay that can be used to evaluate the relative toxicity of solid and liquid media and to evaluate plant tolerance to toxic compounds. Using this method, it is possible to use percent germination, root and shoot growth as parameters. For this bioassay, solid substrate such as soil or acid-washed sand is sandwiched between two layers of microfiber filter paper inside a perforated Petri dish that bathes within a larger reservoir. The addition of distilled water or liquid media to the reservoir causes the liquid to pass through the perforations in the inner dish, through the solid substrate to form a film at the top of the filter paper that covers the substrate. Seeds are placed into the film of solution for germination and subsequent growth. Unlike germinating seeds in soil, the early germination of seeds can easily be observed. A distinction can then be made between seeds that do not germinate and those that do germinate but subsequently cease growth, giving the toxicity bioassay additional sensitivity. Root growth may be monitored as a time-course or measured at the end of the experiment. In addition, visual symptoms of stress such as swelling or coloration can be observed throughout the experiment. While shoot growth is often found to poorly reflect toxicity of media, we have found a good response in this parameter. The bioassay has been used to test relative toxicities of metal-contaminated soils, metal solutions, hydrocarbon-contaminated soils and soil leachates and airborne hydrocarbons accumulated in plant tissues.     

Fractionation of Phenolic Compounds Extracted from Propolis and Their Activity in the Yeast Saccharomyces cerevisiae

We have here investigated the activities of Slovenian propolis extracts in the yeast Saccharomyces cerevisiae, and identified the phenolic compounds that appear to contribute to these activities. We correlated changes in intracellular oxidation and cellular metabolic energy in these yeasts with the individual fractions of the propolis extracts obtained following solid-phase extraction. The most effective fraction was further investigated according to its phenolic compounds.     

Bioactive Neolignans and Other Compounds from Magnolia grandiflora L.: Isolation and Antiplasmodial Activity

Bioassay‐guided fractionation of a methanol extract of Magnolia grandiflora against Plasmodium falciparum yielded two new ( 1 and 2 ) and six known ( 3 – 8 ) bioactive compounds. The structures of the new compounds were assigned by mass spectrometric and 1D‐ and 2D‐NMR data. Known compounds were identified by comparison of ¹H‐NMR and MS data with literature data. The two known neolignans 3 and 4 showed moderate antiplasmodial activity with the IC₅₀ values of 2.8 ± 0.1 and 3.4 ± 0.1 μm , respectively. Weak antiplasmodial activity was recorded for compounds 1 , 2 , 5 , 6 , 7 , and 8 , with the IC₅₀ values of 38 ± 2, 23 ± 2, 16.5 ± 0.2, 86 ± 1, 44 ± 4, and 114 ± 9 μm , respectively.     

Development of an In Vitro Bioassay for Clostridium botulinum Type B Neurotoxin in Foods That Is More Sensitive than the Mouse Bioassay

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin’s light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml⁻¹ (0.5 mouse 50% lethal dose ml⁻¹) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.