• Journal of Life Science
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• v.20 no.5
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• pp.789-793
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• 2010

Artificial chromosome manipulation and amplification of single-copy yeast artificial chromosome (YAC) are usually required in order to use YACs for applications such as physical mapping and functional analysis in eukaryotes. We designed and implemented a Simultaneous YAC Manipulation-Amplification (SYMA) system that combines the copy number amplification system of YAC with a convenient YAC manipulation system. To achieve the desired split and to amplify a YAC clone-harboring plant chromosome, a pBGTK plasmid containing a conditional centromere and thymidine kinase (TK) gene was constructed as a template to amplify the splitting fragment via PCR. By splitting, new 490-kb and 100-kb split YACs containing the elements for copy number amplification were simultaneously generated from a 590-kb YAC clone. The 100-kb split YAC was then successfully amplified 14.4-fold by adding 3 mg/ml sulfanilamide and $50\;{\mu}g/ml$ methotrexate (S3/M50) as inducing substances.

• Molecules and Cells
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• v.37 no.11
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• pp.795-803
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• 2014

To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant, brassinosteroid insensitive1-9 (bri1-9). To identify genes that are antagonistically regulated by ABA and BRs, we examined published Arabidopsis microarray data sets. Of the list of genes identified, those upregulated by ABA but downregulated by BRs were enriched with a BRRE motif in their promoter sequences. After validating the microarray data using quantitative RT-PCR, we focused on RD26, which is induced by salt stress. Histochemical analysis of transgenic Arabidopsis plants expressing RD26pro:GUS revealed that the induction of GUS expression after NaCl treatment was suppressed by co-treatment with BRs, but enhanced by co-treatment with propiconazole, a BR biosynthetic inhibitor. Similarly, treatment with bikinin, an inhibitor of BIN2 kinase, not only inhibited RD26 expression, but also reduced the survival rate of the plant following exposure to salt stress. Our results suggest that ABA and BRs act antagonistically on their target genes at or after the BIN2 step in BR signaling pathways, and suggest a mechanism by which plants fine-tune their growth, particularly when stress responses and growth compete for resources.

• Molecules and Cells
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• v.40 no.3
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• pp.211-221
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• 2017

Transforming growth factor ${\beta}1$ $(TGF{\beta}1)/Smad4$ signaling plays a pivotal role in maintenance of the dynamic balance between bone formation and resorption. The microRNA miR-155 has been reported to exert a significant role in the differentiation of macrophage and dendritic cells. The goal of this study was to determine whether miR-155 regulates osteoclast differentiation through $TGF{\beta}1/Smad4$ signaling. Here, we present that $TGF{\beta}1$ elevated miR-155 levels during osteoclast differentiation through the stimulation of M-CSF and RANKL. Additionally, we found that silencing Smad4 attenuated the upregulation of miR-155 induced by $TGF{\beta}1$. The results of luciferase reporter experiments and ChIP assays demonstrated that $TGF{\beta}1$ promoted the binding of Smad4 to the miR-155 promoter at a site located in 454 bp from the transcription start site in vivo, further verifying that miR-155 is a transcriptional target of the $TGF{\beta}1/Smad4$ pathway. Subsequently, TRAP staining and qRT-PCR analysis revealed that silencing Smad4 impaired the $TGF{\beta}1$-mediated inhibition on osteoclast differentiation. Finally, we found that miR-155 may target SOCS1 and MITF to suppress osteoclast differentiation. Taken together, we provide the first evidence that $TGF{\beta}1/Smad4$ signaling affects osteoclast differentiation by regulation of miR-155 expression and the use of miR-155 as a potential therapeutic target for osteoclast-related diseases shows great promise.

• Molecules and Cells
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• v.40 no.1
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• pp.45-53
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• 2017

Aberrant hypermethylation of Wnt antagonists has been observed in gastric cancer. A number of studies have focused on the hypermethylation of a single Wnt antagonist and its role in regulating the activation of signaling. However, how the Wnt antagonists interacted to regulate the signaling pathway has not been reported. In the present study, we systematically investigated the methylation of some Wnt antagonist genes (SFRP2, SFRP4, SFRP5, DKK1, DKK2, and APC) and their regulatory role in carcinogenesis. We found that aberrant promoter methylation of SFRP2, SFRP4, DKK1, and DKK2 was significantly increased in gastric cancer. Moreover, concurrent hypermethylation of SFRP2 and DKK2 was observed in gastric cancer and this was significantly associated with increased expression of ${\beta}-catenin$, indicating that the joint inactivation of these two genes promoted the activation of the Wnt signaling pathway. Further analysis using a multivariate Cox proportional hazards model showed that DKK2 methylation was an independent prognostic factor for poor overall survival, and the predictive value was markedly enhanced when the combined methylation status of SFRP2 and DKK2 was considered. In addition, the methylation level of SFRP4 and DKK2 was correlated with the patient's age and tumor differentiation, respectively. In conclusion, epigenetic silencing of Wnt antagonists was associated with gastric carcinogenesis, and concurrent hypermethylation of SFRP2 and DKK2 could be a potential marker for a prognosis of poor overall survival.

• Journal of Gastric Cancer
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• v.3 no.2
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• pp.75-79
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• 2003

Purpose: Evidence exists that dysregulation of apoptosis is involved in the pathogenesis of cancer development. The Bcl-$x_{L}$/Bcl-2-associated death promoter (BAD), a member of the Bcl-2 family, is a critical regulatory component of the intrinsic cell-death pathway that exerts its pro-apoptotic effect upon heterodimerization with anti-apoptotic proteins Bcl-2 and Bcl-$X_{L}$. Expression of the BAD protein has been reported in several cancer types, but not in stomach cancer. The aim of this study was to explore the expression status of the BAD protein in gastric carcinomas. Materials and Methods: In the current study, we analyzed the expression of the BAD protein in 60 advanced gastric adenocarcinomas by using immunohistochemistry and a tissue microarray approach. Results: Immunopositivity (defined as $\geq\30\%$) was observed for the BAD protein in 57 ($95\%$) of the 60 cancers. Normal gastric mucosal cells showed weaker expressions of the BAD protein than gastric carcinomas. Conclusion: Taken together, these results suggest that stomach cancer cells in vivo may need BAD protein expression for apoptosis. Also, the higher expression of the BAD protein in stomach cancer cells than in normal gastric mucosal cells suggests that apoptosis might be easily triggered in susceptible stomach cancer cells, thereby producing selective pressure to make more apoptosis-resistant cells during tumor development.

• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1151-1158
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• 2011

In this study, a galactooligosaccharide (GOS) was synthesized using active ${\beta}$-galactosidase (${\beta}$-gal) inclusion bodies (IBs)-containing Escherichia coli (E. coli) cells. Analysis by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry revealed that a trisaccharide was the major constituent of the synthesized GOS mixture. Additionally, the optimal pH, lactose concentration, amounts of E. coli ${\beta}$-gal IBs, and temperature for GOS synthesis were 7.5, 500 g/l, 3.2 U/ml, and $37^{\circ}C$, respectively. The total GOS yield from 500 g/l of lactose under these optimal conditions was about 32%, which corresponded to 160.4 g/l of GOS. Western blot analyses revealed that ${\beta}$-gal IBs were gradually destroyed during the reaction. In addition, when both the reaction mixture and E. coli ${\beta}$-gal hydrolysate were analyzed by high-performance thin-layer chromatography (HP-TLC), the trisaccharide was determined to be galactosyl lactose, indicating that a galactose moiety was most likely transferred to a lactose molecule during GOS synthesis. This GOS synthesis system might be useful for the synthesis of galactosylated drugs, which have recently received significant attention owing to the ability of the galactose molecules to improve the drugs solubility while decreasing their toxicity. ${\beta}$-Gal IB utilization is potentially a more convenient and economic approach to enzymatic GOS synthesis, since no enzyme purification steps after the transgalactosylation reaction would be required.

• Korean Journal of Environmental Agriculture
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• v.30 no.3
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• pp.346-356
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• 2011

BACKGROUND: Chemical fungicides not only may pollute the ecosystem but also can be environmentally hazardous, as the chemicals accumulate in soil. Biological control is a frequently-used environment-friendly alternative to chemical pesticides in phytopathogen management. However, the use of microbial products as fungicides has limitations. This study isolated and characterized a three-antifungal-enzyme (chitinase, cellulase, and ${\beta}$-1,3-glucanase)-producing bacterium, and examined the conditions required to optimize the production of the antifungal enzymes. METHOD AND RESULTS: The antifungal enzymes chitinase, cellulase, and ${\beta}$-1,3-glucanase were produced by bacteria isolated from an sawmill in Korea. Based on the 16S ribosomal DNA sequence analysis, the bacterial strain AM50 was identical to Streptomyces sp. And their antifungal activity was optimized when Streptomyces sp. AM50 was grown aerobically in a medium composed of 0.4% chitin, 0.4% starch, 0.2% ammonium sulfate, 0.11% $Na_2HPO_4$, 0.07% $KH_2PO_4$, 0.0001% $MgSO_4$, and 0.0001% $MnSO_4$ at $30^{\circ}C$. A culture broth of Streptomyces sp. AM50 showed antifungal activity towards the hyphae of plant pathogenic fungi, including hyphae swelling and lysis in P. capsici, factors that may contribute to its suppression of plant pathogenic fungi. CONCLUSION(S): This study demonstrated the multiantifungal enzyme production by Streptomyces sp. AM50 for the biological control of major plant pathogens. Further studies will investigate the synergistic effect, to the growth regulations by biogenic amines and antifungal enzyme gene promoter.

• Mycobiology
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• v.45 no.4
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• pp.362-369
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• 2017

We assessed the regulation of cryparin, a class II hydrophobin, using three representative mitogen-activated protein kinase (MAPK) pathways in Cryphonectria parasitica. Mutation of the CpSlt2 gene, an ortholog of yeast SLT2 in the cell wall integrity (CWI) pathway, resulted in a dramatic decrease in cryparin production. Similarly, a mutant of the CpBck1 gene, a MAP kinase kinase kinase gene in the CWI pathway, showed decreased cryparin production. Additionally, mutation of the cpmk1 gene, an ortholog of yeast HOG1, showed decreased cryparin production. However, mutation of the cpmk2 gene, an ortholog of yeast Kss1/Fus3, showed increased cryparin production. The easy-wet phenotype and accumulation of the cryparin transcript in corresponding mutants were consistent with the cryparin production results. In silico analysis of the promoter region of the cryparin gene revealed the presence of binding motifs related to downstream transcription factors of CWI, HOG1, and pheromone responsive pathways including MADS-box- and Ste12-binding domains. Real-time reverse transcriptase PCR analyses indicated that both CpRlm1, an ortholog of yeast RLM1 in the CWI pathway, and cpst12, an ortholog of yeast STE12 in the mating pathway, showed significantly reduced transcription levels in the mutant strains showing lower cryparin production in C. prasitica. However, the transcription of CpMcm1, an ortholog of yeast MCM1, did not correlate with that of the mutant strains showing downregulation of cryparin. These results indicate that three representative MAPK pathways played a role in regulating cryparin production. However, regulation varied depending on the MAPK pathways: the CWI and HOG1 pathways were stimulatory, whereas the pheromone-responsive MAPK was repressive.

• The Plant Pathology Journal
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• v.27 no.1
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• pp.89-92
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• 2011

Cyclic AMP receptor-like protein (Clp), is known to be a global transcriptional regulator for the expression of virulence factors in Xanthomonas campestris pv. campestris (Xcc). Sequence analysis showed that Xanthomonas oryzae pv. oryzae (Xoo) contains a gene that is strongly homologous to the Xcc clp. In order to determine the role of the Clp homolog in Xoo, a marker exchange mutant of $clp_{xoo4158}$ was generated. Virulence and virulence factors, such as the production of cellulase, xylanase, and extracellular polysaccharides (EPS) and swarming motility were significantly decreased in the $clp_{xoo4158}$ mutant. Moreover, the mutation caused the strain to be more sensitive to hydrogen peroxide and to over-produce siderophores. Complementation of the mutant restored the mutation-related phenotypes. Expression of $clp_{xoo4158}$, assessed by reverse-transcription realtime PCR and clp promoter activity, was significantly reduced in the rpfB, rpfF, rpfC, and rpfG mutants. These results suggest that the clp homolog, $clp_{xoo4158}$, is involved in the control of virulence and resistance against oxidative stress, and that expression of the gene is controlled by RpfC and RpfG through a diffusible signal factor (DSF) signal in Xanthomonas oryzae pv. oryzae KACC10859.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.201-206
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• 1998

To develop the fruits of cucumber (Cucumis sativus L.) producing high yields of superoxide dismutase (SOD), the MnSOD cDNA from pea (Pisum sativum) under the control of the cauliflower mosaic virus 35S promoter was introduced into cucumber using Agrobacterium tumefaciens (strain LBA 4404)-mediated transformation. The kanamycin-resistant shoots were selected on the selection medium containing MS basal salt, 1.0 mg/L zeatin, 0.1 mg/L IAA, 300 mg/L claforan, and 100 mg/L kanamycin. After 6 weeks of culture on the selection medium, the shoots were transferred to MS medium containing 0.2 mg/L NAA to induce roots. PCR analysis using the primers for neomycin phosphotransferase (NPTII) gene revealed that three plantlets were transformed. The fruits of one transgenic plant had approximately 3.2-fold higher SOD activity than those of non-transgenic plants. MnSOD isoenzyme band was strongly detected on native gel in fruits of transgenic plants.