The study compared the juice and wine odorants of the Cheongsoo grape cultivar with those of Chardonnay and Reisling wines. The volatile compounds of the three grape varieties were analyzed using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The most common volatile compounds in the juices from the three cultivars were terpenes, C13-norisoprenoids, etones, alcohols, and aldehydes. Terpenes were established as the most abundant group of volatiles in Cheongsoo grape juice, where as aldehydes predominated in Chardonnay and Riesling juices. Forty-two volatile compounds (acids, alcohols, esters, and others) were detected in the three white wines. The concentration of esters was about four times higher in Cheongsoo wine than in Chardonnay and Riesling wines. Five esters found in the Cheongsoo wine, namely, isoamyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, exhibited high odor activity values (OAV) of >1. Furthermore, only Cheongsoo wine had a high OAV for isoamyl acetate odorant, which is associated with banana and sweet aromas. Therefore, the abundant and varied esters are believed to be key volatile fruity/sweet odorants in Cheongsoo wine.

Background : Despite the presence of various bioactive compounds in ginseng, there is lack of study on the variations of bioactive compounds in ginseng according to the cultivation of soil and the applied fertilizer types (or amount). Therefore, this study aims to examine the variations of 37 fatty acids (FA) and 8 vitamin E (Vit-E) vitamers in 6-year-old ginseng root cultivated in different soil types with different fertilizers regimes. Methods and Results : The profiling of 37 FAs and 8 Vit-E vitamers in 6-year-old ginseng roots was measured by gas chromatography coupled with a flame ionization detector, and then these results were statistically analyzed with chemometrics. The FA and Vit-E content in ginseng roots varied significantly with respect to soil cultivation conditions due to organic fertilizer types and amounts used. Unsaturated FA in ginseng is approximately 2.7 fold higher than the saturated FA. Linoleic, palmitic, and oleic acids were the most abundant FAs found in the ginseng roots. Also, the major Vit-E vitamer found in ginseng root is α -tocopherol. In particular, the application of rice straw compost or food waste fertilizer was increased to create nutritionally-desirable FAs and bioactive Vit-E in ginseng root. In addition, phytonutrient profiling coupled with chemometrics can be used to discriminate the cultivation conditions of ginseng. Conclusion : This preliminary study extends our understanding about the variations of FA and Vit-E in ginseng root depending on cultivation conditions. Hence, these results can be useful as basic information for reliable ginseng production containing high amounts of phytonutrients in a paddy-converted field.

Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs

To understand molecular mechanisms underlying adaptation of plant cells to saline stress and stress memory, we developed Arabidopsis callus suspension-cultured cells adapted to high salt. Adapted cells to high salt exhibited enhanced tolerance compared to control cells. Moreover, the salt tolerance of adapted cells was stably maintained even after the stress is relieved, indicating that the salt tolerance of adapted cells was memorized. Salt-adapted and stress memorized cells were densely aggregated and formed multi-layered cell lump. Cell morphology analysis using transmission electron microscopy indicated that cell wall thickness of salt-adapted cells was significantly induced compared to control cells. In order to characterize metabolic responses of plant cells during adaptation to high salt stress as well as stress memory, we compared metabolic profiles of salt-adapted and stress-memorized cells with control cells by using NMR spectroscopy. A principle component analysis showed clear metabolic discrimination among control, salt-adapted and stress-memorized cells. Compared with control cells, metabolites related to shikimate metabolism such as tyrosine, and flavonol glycosides, which are related to protective mechanism of plant against stresses were largely up-regulated in adapted cell lines. Moreover, coniferin, a precursor of lignin, was more abundant in salt-adapted cells than control cells. The results provide new insight into metabolic level mechanisms of plant adaptation to saline stress as well as stress memory.

To identify novel signaling components involved in regulation of plant responses to phosphate (Pi) starvation, we screened an Arabidopsis T-DNA activation tagging library for mutants with altered Pi-starvation responses. Here, we report the identification and characterization of novel activation-tagged mutant involved in Pi starvation signaling in Arabidopsis. The hpd (hypersensitive to Pi deficiency) mutant exhibits enhanced phosphate uptake and altered root architectural change under Pi starvation compared to wild type. Expression analysis of auxin-responsive DR5::GUS reporter gene in hpd mutant indicated that both auxin biosynthesis and auxin translocation under Pi starvation are suppressed in hpd mutant plants. Impaired auxin translocation in roots of hpd mutant was attributable to abnormal root architecture changes in Pi starvation conditions. Mis-regulation of auxin translocation in hpd mutant was further confirmed by analysis of expression patterns of auxin efflux carrier proteins, PIN-FORMED (PIN) 1, 2, and 3 fused with GFP. Not only expression levels but also expression domains of PIN proteins were altered in hpd mutant in response to Pi starvation. Molecular genetic analysis of hpd mutant revealed that the mutant phenotype is caused by the lesion in ENHANCED SILENCING PHENOTYPE4 (ESP4) gene whose function is proposed in mRNA 3’-end processing. The results propose that mRNA processing plays crucial roles in Pi homeostasis as well as developmental reprograming in response to Pi deprivation in Arabidopsis.

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

Citrus is one of the major fruits produced in Korea. There are about 20 species mainly grown in Jeju Island, Korea. Four representative species, which are quite different in the shape of leaf and the taste of fruit, were selected and were used to profile the transcriptomes. These species are ‘Miyagawa Wase’ (C. unshiu Marcov.) satsuma mandarin, ‘Kiyomi’ (C. unshiu Marcov. × C. sinensis) mandarin hybrid, ‘Dangyuja’ (C. grandis) and ‘Natsudaidai’ (C. natsudaidai). Classification of the up-regulated and down-regulated genes using the Cluster of Orthologous Groups of proteins (COG) database reveals that the number of genes included in each group differed significantly among the four species. Several genes that showed significant differences in expression on the microarray were selected and their expression patterns were examined by reverse transcription- ploymerase chain reaction. Metabolic genes such as tyrosine decarboxylase and β-glucosidase ligase were found to be highly expressed in Miyagawa Wase, relative to other species. On the other hand, the expression level of mannose phosphate isomerase was lower in Miyagawa Wase. An efflux pump gene was found to be up-regulated in Kiyomi, whereas cinnamyl-alcohol dehydrogenase was down-regulated. β-carotene 15,15’-dioxygenase, which is involved in the vitamin metabolism, was up-regulated in Natsudaidai. Interspecific differentiations of gene expression are analyzed in terms of the metabolic pathways and their possible roles in citrus species.

Phosphorus is one of the macronutrients essential for plant growth and development, as well as crop productivity. Many soils around the world are deficient in phosphate (Pi) that plants can utilize. To cope with the stress of Pi starvation, plants have evolved many adaptive strategies, such as changes of root architecture and enhanced Pi acquisition form soil. To understand molecular mechanism underlying Pi starvation stress signaling, we characterized the activation-tagged mutant showing altered responses to Pi deficiency compared to wild type Arabidopsis and named hsp3 (hypersensitive to Pi starvation3). hsp3 mutant exhibits enhanced phosphate transporter activity, resulting in higher Pi content than wild type. However, in root architectural change under Pi starvation, hsp3 shows hyposensitive responses than wild type, such as longer primary root elongation, lower lateral root density. Histochemical analysis using hsp3 mutant expressing auxin-responsive DR5::GUS reporter gene, indicated that auxin allocation from primary to lateral roots under Pi starvation is aborted in hsp3 mutant. Molecular genetic analysis of hsp3 mutant revealed that the mutant phenotype is caused by the lesion in ENHANCED SILENCING PHENOTYPE4 (ESP4) gene whose function is proposed in mRNA 3’ end processing. Here, we propose that mRNA processing plays a crucial role in Pi homeostasis in Arabidopsis.

In order to adapt to various environmental stresses, plants have employed diverse regulatory mechanisms of gene expression. Epigenetic changes, such as DNA methylation and histone modifications play an important role in gene expression regulation under stress condition. It has been known that some of epigenetic modifications are stably inherited after mitotic and meiotic cell divisions, which is known as stress memory. To understand molecular mechanisms underlying stress memory mediated by epigenetic modifications, we developed Arabidopsis suspension-cultured cell lines adapted to high salt by stepwise increases in the NaCl concentration up to 120 mM. Adapted cell line to 120 mM NaCl, named A120, exhibited enhanced salt tolerance compared to unadapted control cells (A0). Moreover, the salt tolerance of A120 cell line was stably maintained even in the absence of added NaCl, indicating that the salt tolerance of A120 cell line was memorized even after the stress is relieved. By using salt adapted and stress memorized cell lines, we intend to analyze the changes of DNA methylation, histone modification, transcriptome, and proteome to understand molecular mechanisms underlying stress adaptation as well as stress memory in plants.