989 resultados para spider toxins


Relevância:

20.00% 20.00%

Publicador:

Resumo:

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins.

H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1–1000 ng/ml), T-2 toxin (0.0005–5 ng/ml) or HT-2 toxin (0.005–50 ng/ml) for 48 h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes.

Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P < 0.05) by DON (100 ng/ml), T-2 toxin (0.5 ng/ml) and HT-2 toxin (5 ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study.

Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 µg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L. © 2012 Elsevier B.V. All rights reserved.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA) and domoic acid (DA), for PSP, DSP and ASP, respectively. In this study a multi-detection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multi-detection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in buffer was similar in single- and multi-detection: 5.6 ± 1.1 ng/mL for STX, 1.1 ± 0.03 ng/mL for OA and 1.9 ± 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90 % for STX, 80 % for OA and 100 % for DA). This microsphere-based multi-detection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Paralytic shellfish poisoning (PSP) is a potentially fatal human health condition caused by the consumption of shellfish containing high levels of PSP toxins. Toxin extraction from shellfish and from algal cultures for use as standards and analysis by alternative analytical monitoring methods to the mouse bioassay is extensive and laborious. This study investigated whether a selected MAb antibody could be coupled to a novel form of magnetic microsphere (hollow glass magnetic microspheres, brand name Ferrospheres-N) and whether these coated microspheres could be utilized in the extraction of low concentrations of the PSP toxin, STX, from potential extraction buffers and spiked mussel extracts. The feasibility of utilizing a mass of 25 mg of Ferrospheres-N, as a simple extraction procedure for STX from spiked sodium acetate buffer, spiked PBS buffer and spiked mussel extracts was determined. The effects of a range of toxin concentrations (20-300 ng/mL), incubation times and temperature on the capability of the immuno-capture of the STX from the spiked mussel extracts were investigated. Finally, the coated microspheres were tested to determine their efficiency at extracting PSP toxins from naturally contaminated mussel samples. Toxin recovery after each experiment was determined by HPLC analysis. This study on using a highly novel immunoaffinity based extraction procedure, using STX as a model, has indicated that it could be a convenient alternative to conventional extraction procedures used in toxin purification prior to sample analysis.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

A novel multiplex microarray has been developed for the detection of five groups of harmful algal and cyanobacterial toxins found in marine, brackish, and freshwater environments including domoic acid (DA), okadaic acid (OA, and analogues), saxitoxin (STX, and analogues), cylindrospermopsin (CYN) and microcystins (MC, and analogues). The sensitivity and specificity were determined and feasibility to be used as a screening tool investigated. Results for algal/cyanobacterial cultures (n = 12) and seawater samples (n = 33) were compared to conventional analytical methods, such as high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Detection limits for the 15 min assay were 0.37, 0.44, 0.05, 0.08, and 0.40 ng/mL for DA, OA, STX, CYN, and MC, respectively. The correlation of data obtained from the microarray compared to conventional analysis for the 12 cultures was r(2) = 0.83. Analysis of seawater samples showed that 82, 82, 70, 82, and 12% of samples were positive (>IC20) compared to 67, 55, 36, 0, and 0% for DA, OA, STX, CYN, and MC, respectively, for conventional analytical methods. The discrepancies in results can be attributed to the enhanced sensitivity and cross-reactivity profiles of the antibodies in the MBio microarray. The feasibility of the microarray as a rapid, easy to use, and highly sensitive screening tool has been illustrated for the five-plex detection of biotoxins. The research demonstrates an early warning screening assay to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying harmful toxins in water samples.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

A huge variety of proteins are able to form fibrillar structures(1), especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand(2,3). Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal(4,5) and carboxy-terminal(6) domains. The N-terminal domain comprises a secretion signal, but further functions remain unassigned. The C-terminal domain was implicated in the control of solubility and fibre formation(7) initiated by changes in ionic composition(8,9) and mechanical stimuli known to align the repetitive sequence elements and promote beta-sheet formation(10-14). However, despite recent structural data(15), little is known about this remarkable behaviour in molecular detail. Here we present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Major ampullate silk fibers of orb web-weaving spiders have impressive mechanical properties due to the fact that the underlying proteins partially fold into helical/amorphous structures, yielding relatively elastic matrices that are toughened by anisotropic nanoparticulate inclusions (formed from stacks of beta-sheets of the same proteins). In vivo the transition from soluble protein to solid fibers involves a combination of chemical and mechanical stimuli (such as ion exchange, extraction of water and shear forces). Here we elucidate the effects of such stimuli on the in vitro aggregation of engineered and recombinantly produced major ampullate silk-like proteins (focusing on structure-function relationships with respect to their primary structures), and discuss their relevance to the storage and assembly of spider silk proteins in vivo. (C) 2009 Elsevier Inc. All rights reserved.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Natural spider silk fibers have impressive mechanical properties (outperforming many man-made fibers) and are, moreover, biocompatible, biodegradable, and produced under benign conditions (using water as a solvent at ambient temperature). The problems associated with harvesting natural spider silks inspired us to devise a method to produce spider silk-like proteins biotechnologically (the first subject tackled in this highlight); we subsequently discuss their processing into various materials morphologies, and some potential technical and biomedical applications.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1 μg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneous detection of the toxins. The IC50's using this method were 1.9 ± 0.1 μg L−1 MC-LR, 1.3 ± 0.1 μg L−1 CYN, 61 ± 4 μg L−1 ANA-a, 5.4 ± 0.4 μg L−1 STX and 4.9 ± 0.9 μg L−1 DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC–IT-TOF-MS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC–IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

The rapid destruction of microcystin, a cyanobacterial toxin, using a titanium dioxide photocatalyst is observed; the process is extremely efficient with high concentrations of toxin completely undetectable within 10-40 min, depending on the initial concentration.

Relevância:

20.00% 20.00%

Publicador:

Resumo:

Cyanobacterial (blue-green algal) toxins are extremely toxic naturally occurring substances which display hepato- and neurotoxic behaviour (1, 2). In this paper we report the application of titanium dioxide photocatalysis for the destruction of two of these compounds, microcystin-LR and anatoxin-a. The destruction of microcystin appears to follow Langmuir-Hinshelwood kinetics although a discrepancy was observed between adsorption constants determined for the photocatalytic process with those obtained from dark isotherms. A square root dependence between illumination intensity and rate of microcystin destruction was noted. When the destruction was performed in the presence of the naturally occurring pigment it appeared that the pigment also contributes to the destruction of the toxin. Toxicity studies on the photocatalysed toxin solutions indicates that the toxicity is substantially reduced within 30 min photolysis.