Microcystin-LR (MCLR) is the most toxic and most frequently encountered hepatotoxin in the aquatic environment. This study investigated the protein profiles of zebrafish (Danio rerio) livers chronically exposed to MCLR concentrations (2 or 20 microg/l) using the proteomic approach as well as cell ultrastructure, protein phosphatase (PP) activity, protein phosphatase 2A (PP2A) abundance, and toxin content analysis of the hepatic tissue. The results showed that, after 30-day exposure, the presence of MCLR strikingly enhanced toxin accumulation and the PP activity in zebrafish livers. However, the PP2A amounts were independent of toxin treatments. MCLR caused a noticeable damage to liver ultrastructure, a widespread swelling in the rough endoplasmatic reticulum and mitochondria was observed in the MCLR-exposed hepatocytes, and a honeycomb-like structure was formed in the treated nucleoli. Comparison of two-dimensional electrophoresis (2-DE) protein profiles of MCLR-exposed and nonexposed zebrafish livers revealed that the abundance of 22 proteins, measured by 2-DE, was remarkably altered in response to toxin exposure. These proteins were involved in cytoskeleton assembly, macromolecule metabolism, oxidative stress, and signal transduction, indicating that MCLR toxicity in fish liver is complex and diverse. Thus, proteomics provides a new insight into MCLR toxicity, that chronic toxicity of MCLR is different from acute toxicity, and we speculate that the reactive oxygen species pathway might be the main toxic pathway instead of the PP one. Moreover, even a low concentration of MCLR in water could significantly interrupt cellular processes, and more care should be taken in determining the criterion for MCLR content in drinking water.
A photorefreshable and photoenhanced electrochemical sensing platform for bisphenol A (BPA) detection based on Au nanoparticles (NPs) decorated carbon doped TiO2 nanotube arrays (TiO2/Au NTAs) is described. The TiO2/Au NTAs were prepared by quick annealing of anodized nanotubes in argon, followed by controllable electrodeposition of Au NPs. The decoration of Au NPs not only improved photoelectrochemical behavior but also enhanced electrocatalytic activities of the resulted hybrid NTAs. Meanwhile, the high photocatalytic activity of the NTAs allowed the electrode to be readily renewed without damaging the microstructures and surface states after a short UV treatment. The electrochemical detection of BPA on TiO2/Au NTAs electrode was significantly improved under UV irradiation as the electrode could provide fresh reaction surface continuously and the further increased photocurrent resulting from the improved separation efficiency of the photogenerated electron-hole pairs derived from the consumption of holes by BPA. The results showed that the refreshable TiO2/Au NTAs electrode is a promising sensor for long-term BPA monitoring with the detection limit (S/N = 3) of 6.2 nM and the sensitivity of 2.8 μA·μM(-1)·cm(-2).
A comprehensive study to find the optimal sample preparation conditions for two-dimensional electrophoresis (2-DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2-DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre-electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2-DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2-DE but this treatment did yield 2-DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2-DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3-[(3-cholamidoprphyldimethylamino]-1-propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2-DE analysis on the soluble protein fractions extracted from light-starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2-DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.
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