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In this study, we describe an effective visualizing technique for proteins in SDS-PAGE based on the organic dye, Eosin B, the sensitivity of which can be further strengthened by the addition of magnesium to the staining solution after electrophoresis. The newly developed protocol is low in cost and easily performed compared with the common methods for protein analysis in 1-D and 2-D gels. It provides a much better sensitivity (0.2 ng of single protein band) than that of imidazole-zinc negative stain for fixing and staining within 1 h, and an excellent performance in terms of compatibility with MALDI-TOF MS. The results show that similar identification scores and numbers of matched peptides were obtained by both methods. Furthermore, the effects of different metal salts on the quality of protein visualization by Eosin B were also investigated. Because of its sensitivity, stability, and safety, this stain may be a more practical method for protein determination in the routine laboratory.

A negative detection method for proteins on SDS-PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water-soluble protein-dye complex. Negative staining of proteins using EY, allows high-sensitivity, low-cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole-zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high-throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.

One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. In this study, we found that FGF-21 can significantly attenuate ischemia-reperfusion (I/R) induced damage in H9c2 cells (rat heart). However, it is unclear which signal transduction pathway is involved in the cardioprotective effect of FGF-21. Thus, this study was designed to investigate the potential mechanism induced by FGF-21. The results showed that FGF-21 treatment prevented the oxidative stress and apoptosis associated with I/R damage by reducing the levels of superoxide anions, inhibiting glycogen synthase kinase (GSK) 3β by activating Akt phosphorylation, and recovering the levels of ATP synthase pyruvate kinase isozymes M1 and protein kinase C, thereby improving energy supply. In summary, we conclude that FGF-21 protects H9c2 cells against I/R injury mainly through the Akt-GSK-3β-caspase-3 dependent pathway, preventing oxidative stress, and recovery of the energy supply. [ABSTRACT FROM AUTHOR]

Abstract: Mycotoxins are commonly encountered natural products, and are capable of poisoning animals or humans that inhale mold particles from mycotoxin-contaminated foods. Ochratoxin A (OTA) is produced by Aspergillu ochracus and Penicillium verrucosum, and is often found in cereals and agricultural products. Although previous studies have focused on the potent nephrotoxicity and renal carcinogenicity of OTA, more recent studies suggest that it accumulates in the brain and causes oxidative stress and DNA damage in various brain regions and neuronal populations. In the present study, we undertook to investigate the potential harm caused by environmental exposure to OTA in terms of its effects on neuronal cell viability and proteome profiles. OTA was found to significantly reduce the viabilities of human neuroblastoma SH-SY5Y and mouse hippocampal HT22 cells, as assessed by lactic dehydrogenase release into culture media. Generation of reactive oxygen species was detected in OTA-treated SH-SY5Y and HT22 cells, however, caspase activation and increase in p53 phosphorylation were only detected in HT22 cells, and the expressions of several proteins were found to be significantly altered after treating HT22 cells with OTA. Valosin containing protein, prolyl 4-hydroxylase, Atp5b protein, nucleophosmin 1, eukaryotic translation elongation factor 1 delta isoform, ornithine aminotransferase, prohibitin, and peroxiredoxin 6, which have been suggested to be implicated in the pathogenesis of neurodegenerative disorders, were up-regulated. Our findings suggest that coordinated regulations of molecular networks are involved in the OTA-induced cytotoxicity and that proteome response can be an indicative for neurodegeneration. [Copyright &y& Elsevier]

In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1–0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches. [ABSTRACT FROM AUTHOR]

Abstract: A convenient silver staining method for protein in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2ng/band. [Copyright &y& Elsevier]