Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis.

ObjectivesGlucagon-like peptide-1 (GLP-1) is an intestinally secreted hormone and it plays an important role in the regulation of glucose homeostasis. However, the possible role of GLP-1 in the differentiation of adipose-derived stem cells (ADSCs) remains unknown. Therefore this study investigated the effect of GLP-1 on the differentiation of ADSCs into osteoblasts and adipocytes.MethodsADSCs were isolated from human adipose tissues of the abdomens, cultured and characterized by flow cytometry and multi-lineage potential assay. ADSCs were induced in osteogenic and adipogenic media treated with two different doses (10 and 100 nM) of GLP-1, and then the effect of GLP-1 on differentiation of ADSCs into osteoblast and adipocyte was examined. The signaling pathway involved in these processes was also examined.ResultsIsolated human ADSCs expressed mesenchymal stem cell (MSC) specific markers as well as GLP-1 receptor (GLP-1R) proteins. They also showed multiple-lineage potential of MSC. GLP-1 was upregulated the activity and mRNA expression of osteoblast-specific marker, alkaline phosphatase and the mineralization of calcium. In contrast, GLP-1 significantly suppressed the expression of adipocyte-specific markers, peroxisome proliferator-activated receptor gamma (PPAR-γ), lipoprotein lipase (LPL) and adipocyte protein 2 (AP2). This decreased expression of adipocyte specific markers caused by GLP-1 was significantly reversed by the treatment of extracellular signal-regulated kinase (ERK) inhibitor, PD98059 (P < 0.05).ConclusionThis result demonstrates that GLP-1 stimulates osteoblast differentiation in ADSCs, whereas it inhibits adipocyte differentiation. The ERK signaling pathway seems to be involved in these differentiation processes mediated by GLP-1.

High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates.

Abstract. Traditional medicinal plants have been used in the treatment of various diseases for centuries. A number of plant-derived compounds have been proposed as anticancer agents and are currently undergoing medical development. Petasites japonicus (PJ), also known as Butterbur, is a herb cultivated in East Asia that is used as a traditional herbal medicine. The aim of the present study was to investigate whether a methanol extract of PJ demonstrated anticancer activity against Hep3B hepatocellular carcinoma (HCC) cells. The anticancer property and underlying mechanism of the extract were evaluated by assessing the effect on cell viability, nuclear morphology and the expression of phosphorylated (p)-mTOR, p-Akt, β-catenin and p-glycogen synthase kinase-3β, which are markers for cancer cell proliferation and metastasis. These results were obtained by the MTT assay, fluorescence microscopy and Western blot analysis. The methanol extract of PJ was shown to decrease the cell viability in a concentration-dependent manner. In addition, the methanol extract of PJ was found to inhibit the growth of Hep3B HCC cells through inhibiting the Akt/mTOR and Wnt signaling pathways. These results suggest that the methanol extract of PJ exerts an anticancer effect on Hep3B HCC cells.

No abstract