Although Sophorae Flos (SF) has been reported to exert an anti-cancer activity, molecular targets and mechanisms associated with anti-cancer activity of SF have been unclear. Because cyclin D1 has been regarded as an important regulator in the cell proliferation, we focused cyclin D1 and investigated the effect of SF on the cyclin D1 regulation in light of elucidating the molecular mechanism for SF’s anti-cancer activity. The treatment of SF decreased cellular accumulation of cyclin D1 protein. However, SF did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated SF-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with SF. In addition, a point mutation of threonine-286 to alanine attenuated SF-mediated cyclin D1 downregulation. Inhibition of ERK1/2 by a selective inhibitor, PD98059 suppressed cyclin D1 downregulation by SF. From these results, we suggest that SF-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via ERK1/2. SF-induced proteasomal degradation of cyclin D1 might inhibit proliferation in human colorectal cancer cells. The current study provides information on molecular events for an anti-cancer activity of SF

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.

The flower buds of Sophora japonica L (SF), as a well-known traditional Chinese medicinal herb, have been used to treat bleeding-related disorders such as hematochezia, hemorrhoidal bleeding, dysfunctional uterine bleeding, and diarrhea. However, no specific anti-cancer effect and its molecular mechanism of SF have been described. Thus, we performed in vitro study to investigate if treatment of SF affects activating transcription factor 3 (ATF3) expression and ATF3-mediated apoptosis in human colorectal cancer cells. The effects of SF on cell viability and apoptosis were measured by MTT assay and Western blot analysis against cleaved poly (ADP-ribose) polymerase (PARP). ATF3 activation induced by SF was evaluated using Western blot analysis, RT-PCR and ATF3 promoter assay. SF treatment caused decrease of cell viability and increase of apoptosis in a dose-dependent manner in HCT116 and SW480 cells. Exposure of SF activated the levels of ATF3 protein and mRNA via transcriptional regulation in HCT116 and SW480 cells. Inhibition of extracellular signal-regulated kinases (ERK) 1/2 by PD98059 and p38 by SB203580 attenuated SF-induced ATF3 expression and transcriptional activation. Ectopic ATF3 overexpression accelerated SF-induced cleavage of PARP. These findings suggest that SF-mediated apoptosis may be the result of ATF3 expression through ERK1/2 and p38-mediated transcriptional activation.

UGT72E3/2 gene encodes UDP-glycosyltransferase shown to glucosylate several phenylpropanoids such as syringin and coniferin. Syringin has effect of anti-stress and anti-fatigue. Korean soybean variety Kwangan was transformed with UGT72E3/2 gene. This gene was transformed into Kwangan using highly efficient soybean transformation system. This study used two promoters, beta-conglycinin promoter for seed-specific expression and 35s promoter for total expression. Transgenic plants were confirmed for gene introduction and their expression using PCR and RT-PCR. The analysis of syringin in transgenic plants was performed using HPLC. Currently, the confirmation of stable gene introduction with UGT72E3/2 gene is also performing by Southern blot analysis.

Soybean mosaic virus (SMV), a member of Potyviridae family, is one of the most typical viral diseases and results in yield and quality loss of cultivated soybean. Due to the depletion of genetic resources for resistance breeding, a trial of genetic transformation to improve disease resistance has been performed by introducing SMV-CP and HC-Pro gene by RNA interference (RNAi) method via Agrobacterium-mediated transformation. Transgenic plants were infected with SMV strain G5 and investigated the viral response. As a result, two lines (3 and 4) of SMV-CP(RNAi) transgenic plants and three lines (2, 5 and 6) of HC-Pro(RNAi) transgenic plants showed viral resistance. In genomic Southern blot analysis, most of lines contained at least one T-DNA insertion in both SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. Subsequent investigation confirmed that no viral CP and HC-Pro gene expression was detected in two SMV-resistant lines of SMV-CP(RNAi) and three lines of HC-Pro(RNAi) transgenic plants, respectively. On the other hand, non-transgenic plants and other lines showed viral RNA expression. Viral symptoms affected seed morphology, and clean seeds were harvested from SMV-resistant line of SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants. In addition, strong viral gene expression was detected from seeds of SMV-susceptible non-transgenic plants and SMV-susceptible transgenic lines. When compared the viral resistance between SMV-CP(RNAi) and HC-Pro(RNAi) transgenic plants, soybean transgenic plants with the HC-Pro gene using RNAi strategy showed much stronger and higher frequency of viral resistance.

Bacillus thuringiensis(Bt) crystal protein (Cry1Ac) genes encode insecticidal δ-endotoxins that are widely used for the development of insect-resistant crops. Common soybean is a crop of economic and nutritious importance in many parts of the world. Korea soybean variety Kwangan was transformed with Bacillus thuringiensis(Bt) crystal protein genes. We transformed three difference Cry1Ac (Cry1Ac and two modified Cry1Ac) genes into Kwangan using highly efficient soybean transformation system. Transgenic plants with Bt crystal protein genes were confirmed for gene introduction and their expression using PCR, real-time PCR, and RT-PCR. We generated 30 independent lines of transgenic soybean plants. Analysis of the flanking sequences isolated by Inverse PCR revealed complex T-DNA insertion patterns and preferential integration of T-DNA into the intergenic spacer region of the soybean genome. We found 5 different intergenic transgenic soybean lines of soybean genome. Currently, the confirmation of stable gene introduction with Bt genes is also performing by southern blot analysis, physiology test, and agronomic characters are investigating.

Our study is performed to confirm the level of genetic diversity and population structure with 80 maize inbred lines (40 waxy inbred lines and 40 flint inbred lines) and to explain the genetic basis of agronomic traits using an association mapping. The 200 SSR loci are confirmed a total of 1,610 alleles in total 80 maize inbred lines. The average number of alleles per locus was 8.05. The average GD was 0.72. The average PIC value was 0.68. The average MAF was 0.40. Population structure was revealed for K=2. Total 80 maize inbred lines were divided by groups I, II and admixed group. The 14 waxy inbred lines were assigned to group I. The 45 inbred lines include 5 waxy inbred lines and 40 flint inbred lines were contained to group II. The 21 waxy inbred lines were contained in the admixed group with lower than membership threshold 0.8. Association mapping between 200 SSR markers and 10 phenotypic traits of waxy/flint maize inbred lines were performed by Q GLM and Q+K MLM. In significant level at 0.01, 72 SSR markers were associated with 10 phenotypic traits using Q GLM. The 4 marker-trait association were detected in Q+K MLM. The results derived from this study will be used for designing efficient new maize breeding programs.

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.

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.

Korean soybean variety Kwangan was transformed with coat protein (CP), helper component-proteinase (HC-Pro), and ABRE binding factor 3 (ABF3) genes using highly efficient soybean transformation system. Among these genes, CP and HC-Pro were transformed using RNAi technology. Transgenic plants with CP were confirmed for gene introduction and their expression using PCR, real-time PCR, RT-PCR, Southern blot, and Northern blot. To investigate the response of viral infection with CP, T1 plants were inoculated with SMV-infected leaves and confirmed the existence of mosaic symptom in both leaves and seeds. Two transgenic lines with CP were highly resistant to SMV with clear leaves and seeds while SMV-susceptible lines showed mosaic symptom with seed mottling. The transcript levels of T1 plants with CP were also determined by northern blot, suggesting that SMV-resistant T1 plants did not show viral RNA expression whereas SMV-susceptible T1 plants showed viral RNA expression. Currently, the response of viral infection with HC-Pro is investigating to produce SMV-resistant soybean transgenic plants, and the physiological experiment with ABF3 is also carrying out to produce drought-tolerant soybean transgenic plants.

Wild rice might have previously unidentified genes important for disease resistance and stress tolerance in response to biotic and abiotic stresses. A set of subtractive library was constructed both from leaves of wild rice plants, Oryza grandiglumis (CCDD, 2n=48), treated with fungal elicitor and from wounded leaves. A partial fragment that was homologous to PR10 genes from other plant species was identified via suppression subtractive hybridization and cDNA macroarray. The obtained full-length cDNA sequence (OgPR10) contains an open reading frame of 480 bp nucleotide, encoding 160 amino acids with a predicted molecular mass of 16.944 kDa and an isoelectric point (pI) of 4.91. The multiple alignment analyses showed the higher sequence homology of OgPR10 with PR10 genes identified in rice plants at amino acid level. The OgPR10 mRNA was not expressed by treatment with wounding, jasmonic acid, and salicylic acid, but markedly expressed in leaves treated with protein phosphatase inhibitors cantharidin and endothall, and yeast extract. In addition, the expression of OgPR10 mRNA was induced within 72 h after treatment with probenazole, one of well-known chemical elicitors, and reached the highest level at 144 h. Heterologous expression of OgPR10 caused growth inhibition and seedling lethality in E. coli and Arabidopsis, respectively. Chemically induced OgPR10 expression with glucocorticoid-mediated transcriptional induction system further reconfirmed its lethality on Arabidopsis seedling. In addition, OgPR10-expressing rice plants, Oryzae sativar were resistant against the infection of rice blast fungus, Magnaporthe grisea. These results indicate that OgPR10 is involved in probenazole- and microbe associated molecular patterns-mediated disease resistance responses in plants and is a potential gene for developing disease resistance crop plants.