Background : Adenophora triphylla var. japonica Hara. is used to cure internal organ, which is called to important one of the five “Sam (ginseng)” oriental medicinal herbs included Panax ginseng, Salvia miltiorrhiza, Sophora flavescens, Scrophularia buergeriana. Korea imported 24 ton 129 thousand dollar for medicinal herbs and 23 ton 29 thousand dollar for food at 2013. This study was carried out to investigate the effect of several organic compost on the growth and root yield of A. triphylla var japonica with organic fertilizer. Methods and Results : Seeds were collected from Adenophora triphylla var. japonica Hara. at last year in nursery of Department of Herbal Crop Research, NIHHS, RDA. Four types organic fertilizer (germiculture (GG), granular mix oil cake (CJ), mixed organic (DJ), plant oil cake (TG)), chemical fertilizer (CF) and non-fertilizer (NF) were treated. It was fertilized on soil before 2 month of planting. Seeds A. triphylla var. japonica were sowed in tray at early March and then were grown during 2 month. It were planted in furrowed nursery at early with 30 × 10 cm planting distance and black vinyl covering condition. Soil chemical property, survival rate, aerial part and root growth characteristics and yield were investigated at harvest time. For main composition analysis, 10 mg of standard (b-sitosterol, lupenone) was taken, 80% MeOH was added to dissolve in 10 ㎖ flask. 0.5, 1, 2, 4 ㎖ was taken from dissolved solution and then was added to 10 ㎖ of 80% MeOH. It was filtered through 0.45 ㎛ filter and then 5 μℓ was taken to make standard solution. Analytical calibration curve was measured to diluted solution within sample concentration. 2 g of ground sample was taken, 40㎖ of 80% MeOH was added to extract by ultrasound sonication during 60 minutes and then it was filtered through 0.2 ㎛ filter to analyze component content. Conclusion : GG and DJ were the most suitable for organic cultivation of denophora triphylla var. japonica. Content of beta-sitosterol showed the highest value in DJ treatment and the lowest value in CF. Content of lupenone showed the highest value in DJ and the lowest value in CJ.

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

The GA application on grapevines induces parthenocarpy, fruit set without fertilization, and the inhibition of pollen tube growth. But the molecular mechanism underlying this inhibition is not understood. Similar defective pollen tube growth within the transmitting tract has been reported in the mutant of GABA transaminase (GABA-T), referred to as pollen-pistil-interaction2 (pop2) in Arabidopsis. In spite of the similarity of pollen tube growth inhibition observed in GA-applied grapevines with that of pop2, only the effects of GABA on stress responses in grapevines have been reported. In present study, transcriptional changes of Vitis GABA metabolic genes, together with changes in GABA levels with or without GA application were analyzed to define how GA application restrained the pollen tube growth in grapevines. A GA solution (Dongbu, Seoul, Korea) at 100 ppm was onto inflorescence clusters 14 days before full bloom (DBF) and clusters were harvested at 0, 1, 2, 4, 7, 9, 12, 14, 16, and 19 days after GA application. Harvested inflorescence samples were immediately frozen in LN2 and extracted RNA and amino acid. The GABA contents were analyzed using high-performance liquid chromatography (Agilent 1100 HPLC, Agilent Technologies, Inc., Santa Clara, USA) equipped with a C18 column (4.6 mm×150 mm, 3.5 μm/VDS optilab, Berlin, Germany), according to the manufacturer’s instructions. Without GA application, the simultaneous high expressions of VvGAD1, VvGAD4 and VvGABA-T2 during 10 to 5 days before full bloom (DBF) showing the activation of GABA metabolism. But the contents of GABA were low before 2 DBF, and it peaked only at near full bloom when expression levels of VvGABA-T2 remained low. After GA application, the contents of GABA were constant during 10 to 5 DBF, although transcription levels of both VvGAD1 and VvGABA-T2 rapidly declined less than 30% of the levels observed without GA application. However, the GABA levels increased more than 2-fold only at near full bloom, compared to those without GA application, and at that time, expression levels of VvGAD1 up-regulated more than 3-fold and those of VvGABA-T2 kept low. But other amino acid contents did not show significant changes. In case of VvSSAHDs, their transcriptional changes with or without GA application were not correlated with GABA levels. These results indicates that GABA levels before pollination is tightly regulated, but GA application alters the GABA-shunt to accumulate excess GABA more than needed for proper pollen tube growth at full bloom. Gibberellin application alters the GABA-shunt to accumulate excess GABA resulting in inhibition pollen tube growth in grapevines.

In this study, enhancement of phosphorus and nitrogen recovery efficiency from livestock manure through Magnesium Ammonium Phosphate (MAP) crystallization method has been suggested as an alternative to solve the problems of the existing phosphorus resource recovery method. It can become a useful fertilizer. This study focused on improvement of phosphorus resource recovery by changing energy density of ultrasonic dose for MAP crystallization. Solubilization rate (as phosphate/phosphorus) of phosphorus in livestock manure was measured by ultrasonic treatment. The energy density range of 100-50,000 of ultrasonic dose was determined. Optimal ultrasonic energy density was 1,000 dose as 64.5% of phosphate ratio. However, when the higher than 1,000 dose of ultrasonic energy density did not more improve phosphate solubilization ratio. Consequently, when use ultrasonic treatment at 1,000 dose of energy density, the phosphorus could recover approximately 65% from livestock manure by MAP crystallization. Moreover, this MAP becomes more valuable due to its nature as a slow-release fertilizer.

This study was carried out to investigate the effects of the Cirsium japonicum var. ussuriense (C. japonicum)extract on serum level of hormones from induced osteoporosis by ovariectomized rats. Two month-old rats were ovariecto-mized (OVX), remained untreated for 8 weeks, and were subsequently administered C. japonicum (200㎎/㎏) every day for8 weeks. We examined the effects of treated C. japonicum on ovariectomy-related changes in Insulin-like Growth Factors(IGF), Insulin-like Growth Factor binding protein-3 (IGBF-3), Estrogen, Calcium, and Phosporus. After 8 weeks, the serumlevels of IGF-I, -II, and IGFBP-3 were higher presented as compared to the other two groups (P<0.05), in the C. japonicumextract treatment on OVX rats. There were differences between OVX and C. japonicum extract treated OVX rats in serumlevels of Ca2+, but Ca2+ levels for the normal group was higher than for the other two groups. The C. japonicum extractincreased both serum IGFs and IGFBP-3 levels on induced osteoporotic rat by ovariectomized. Thus, these results revealedthat the C. japonicum extract is a possible role for improvement of osteoporosis induced-ovariectomized rats and has a greatpotential as an alternative tool for the treatment of osteoporosis.

The patent ductus arteriosus (PDA) is a vascular structure connecting the proximal descending aorta to the roof of the main pulmonary artery, near the origin of the left branch pulmonary artery. Transcatheter closure has become the treatment of choice for most cases of PDA in both children and adults; however, measurement of the exact size and morphology of the shunt in adult cases using only contrast fluoroscopy is difficult. We report on a case of a 49-year-old woman who underwent transcatheter closure of PDA with intravascular ultrasound (IVUS) guideance. In the current case, IVUS is feasible and helpful for measuring the exact size and shape of the PDA.

We investigated the effect of light spectra on circadian rhythm by exogenous prolactin (PRL) by using light emitting diodes (LEDs): red, green, and purple. We injected PRL into live fish or treated cultured brain cells with PRL. We measured changes in the expressions of period 2 (Per2), cryptochrome 1 (Cry1), melatonin receptor 1 (MT1) mRNAs, and MT1 proteins, and in the plasma PRL, serotonin, and melatonin levels. After PRL injection and exposure to green LED light, MT1 expression and plasma melatonin levels were significantly lower, but the expressions of Per2 and Cry1 were significantly higher than others. Plasma serotonin after PRL injection and exposure to red LED light was significantly lower than others. These results indicate that injection of high concentration PRL inhibits melatonin, and inhibited melatonin regulates circadian rhythm via clock genes and serotonin. Thus, exogenous PRL regulates the circadian rhythm and light spectra influence the effect of PRL in goldfish.

The tight regulators of fruit set initiation, gibberellin (GA) and auxin, have been applied for decades to induce parthenocarpy, fruit set without fertilization. The integration of GA and auxin signaling mediated by either GA or auxin application during parthenocarpy has been actively reported in tomato, and recently we reported that GA application at pre-bloom also activating auxin signaling and down-regulated negative regulators of fruit set initiation in grapevines. However, the activation of auxin signaling upon GA application without up-regulation of auxin biosynthesis is still unclear. In this study, expression patterns of three auxin efflux transporter genes, VvPIN1a, VvPIN2 and VvPIN4, were monitored during inflorescence development in ‘Tamnara’ grapevines with or without GA application. Without GA application, transcription levels of VvPIN1a and VvPIN4 gradually increased from 14 days before full bloom (DBF) to 2 and 5 days after full bloom (DAF), respectively, except down-regulation of VvPIN1a during 5 DBF to full bloom. However, VvPIN2 expression declined steadily after peaking at 10 DBF. With GA application, VvPIN1a did not show significantly different expression patterns when compared to no GA application, with the exception of 4-fold up-regulation at full bloom, but transcription of VvPIN4 was reduced between 5 and 2 DBF. In addition, VvPIN2 was down-regulated between 12 and 10 DBF by more than 50% compared to levels in the absence of GA application. These reductions of both VvPIN2 and VvPIN4 with GA application prior to pollination suggest that GA application might regulate auxin distribution, instead of auxin biosynthesis, to activating auxin signaling during parthenocarpic fruit initiation.

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.

Gibberellic acid (GA) is a well-characterized plant hormone, which plays a critical role in various plant growth and development. including stem elongation, floral indcution and seed development. GA is known to cause enlargement of ripening fruits and, especially in grapevines, GA shows a unique function: the induction of seedlessness in seeded grape varieties. However, despite extensive previous studies about GA, there has been no clear verification of the mechanism that induces seedlessness in grapes. To understand how GA treatment results in artificial parthenocarpy of seeded grapes at molecular levels, we analyzed transcriptional changes in seeded grapes with and without GA application in various inflorescence developmental stages using RNA-seq. At 14 days before flowering (DBF), seeded grapes were treated with 100 ppm GA and clusters were collected at three developmental stages: 7 DBF, full bloom, and 5 days after flowering (DAF). Of a total of 28,974 genes that were mapped to grape genome reference sequences, 7,013 and 9,064 genes were up- and down-regulated, respectively, in the GA-treated grape as compared to the non-GA-treated control at 7 DBF, full bloom, and 5 DAF. Clustering analysis revealed that these genes could be grouped into 9 clusters with different expression patterns. We also carried out functional annotation based on gene ontology categories. There were significant differences in the expression of the GA and auxin-related gene families. These findings expand our understanding of the complex molecular and cellular mechanisms of GA-induced parthenocarpy of grapes and provide a foundation for future studies on seed development in grapevines.

JAK2 V617F mutation is a common event in chronic myeloproliferative disorders. However, de novo acute myeloid leukemia with JAK2 V617F is rarely encountered. The authors report the case of a 74-year-old male with de novo acute myeloblastic leukemia without maturation (AML M1) and a JAk2 V617F heterozygotic mutation. Despite treatment with standard AML regimens, the patient died 2 months after a diagnosis of acute leukemia. This case of an AML patient with a JAK2 V617F mutation with a poor prognosis suggests that despite its rarity, a JAK2 V617F mutational study be considered for prognostic purposes in AML.

Twenty two common millet (Panicum miliaceum L.) varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences with a hope to provide the basic information on their exact origin. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS. Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragments, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region. Amplified DNAs of 400 bp fragment were subcloned and used for further investigation. The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. And critically different areas with insert or deletion were also confirmed. Those sequence difference seems to be used for discrimination of cultivars from different origin and use as molecular markers for origin identification. In phylogenic tree construction, the clear classification was shown where the genotypes from China and Russia is positioned together and stay away from domestic genotypes.

Compared to wide ranges of genetic variation of natural populations, very limited Miscanthus cultivar has been released. This study was the first report on the development of Miscanthus cultivar by means of radiation breeding. Seeds of M. sinensis were initially exposed to gamma rays of 250 Gy for 24 h, generated from a 60Co gamma-irradiator. The irradiated seeds were sown and then the highly tiller-producing mutants were selected for this study. Biomass-related parameters including tiller number, plant height, stem diameter, and leaf number were measured. Ploidy level and internal transcribed spacer (ITS) were investigated to characterize the mutants compared to wild type (WT) Miscanthus. Plant height and tiller number were negatively related, where multi-tillering mutants were relatively short after 4 month growth. However stem diameter and leaf number were greater in mutants. All the materials used in this study were diploid, implying that the mutants with greater tiller numbers and stem diameter were not likely related to polyploidization. Based on the sequence of ITS regions, the mutants demonstrated base changes from the gamma irradiation where G+C content (%) was decreased in the ITS1, but increased in ITS2 when compared to WT sequence. ITS2 region was more variable than in ITS1 in the mutants, which collectively allows identification of the mutants from WT. Those mutants having enhanced tillers and allelic variations might be used as breeding materials for enhanced biomass-producing Miscanthus cultivars.

Understanding the host defense mechanisms in response to brown leaf spot disease caused by Cochliobolus miyabeanus is very important for production of resistant plant. In this study, two-dimensional gel electrophoresis (2-DGE) in conjunction with mass spectrometry was utilized to unravel changes of stress inducible proteins in rice leaves infected with C. miyabeanus. For this purpose, we firstly observed disease developmental process of C. miyabeanus in rice using trypan blue, anilin blue, acid fuchsin staining, and DAB staining for ROS detection and expressional abundance of ROS related proteins in rice leaves inoculated was confirmed by Western blotting. Proteins were extracted by PEG fractionation and their expression patterns were analyzed by 2-DGE and subjected to image analysis using the ImageMaster 6.0 2D Platinum software, resulting in the identification of 86 differentially expressed protein spots with significantly changes (p<0.05) compared with control. MALDI-TOFTOF-MS analysis revealed that 69 proteins including 42 and 27 significantly up- and down-regulated proteins, respectively, were identified. Based on gene ontology analysis, identified proteins were classified according to their functional groups: metabolism (20%), oxygen-detoxifying (13%), protein stress/defense (24%). Thus, these results for the first time suggest that differentially induced proteins may play a key role for understanding host defense mechanisms during rice -C. miyabeanus interaction.

The aim of this study was to evaluate 30 phenolic compounds in adzuki bean germplasm. Adzuki 21653 had the highest content of total phenolics compounds (6597~;~mug~;g-1 ) while 104372 had the lowest concentration. The average total phenolic content of Japanese (2432~;~mug~;g-1 ) adzuki beans was higher than that of Korean (2256~;~mug~;g-1 ) adzuki beans. The average total phenolic contents were 2507~;~mug~;g-1 in small sized adzuki beans from Japan and 2459~;~mug~;g-1 in those from Korea. In large sized adzuki beans, the average total phenolic contents were 1315~;~mug~;g-1 in Japanese seeds and 1232~;~mug~;g-1 in Korean seeds. The average total phenolic contents in medium seeds were 2369~;~mug~;g-1 in Japanese adzuki beans and 1397~;~mug~;g-1 in Korean ones. In small seeds, the total phenolic contents of adzuki beans varied from 524~;~mug~;g-1 to 6597~;~mug~;g-1 in Japanese ones and from 375~;~mug~;g-1 to 6569~;~mug~;g-1 in Korean ones. Japanese and Korean adzuki beans were divided into landraces and wild adzuki beans. In this study, the wild adzuki beans showed higher contents of total phenolics than the native varieties. Specifically, the wild adzuki beans from Korea had the highest concentration of phenolics (3403~;~mug~;g-1 ). All adzuki bean germplasms were measured for their color and were classified into four groups accordingly: A; L < 30, +a, +b; B; L < 30, +a, -b, C; L > 50, +a, +b, D; L > 50, +a, -b. Especially, group B had the highest concentration of total phenolic compounds (2827~;~mug~;g-1 ), whereas group C had the lowest concentration (1882~;~mug~;g-1 ).