• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.196-196
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• 2017

Oilseed crop Camelina (Camelina sativa L.) is a suitable for biodiesel production that has high adaptability under low-nutrient condition like marginal land and requires low-input cost for cultivation. Enhanced abiotic stress tolerance of Camelina is very important for oil production under the wide range of different climate. CsRCI2s (Rare Cold Inducible 2) are related proteins in various abiotic stresses that predicted to localized at plasma membrane (PM) and endoplasmic reticulum (ER). These proteins are consist of eight-family that can be divided into tail (CsRCI2D/E/F/G) and no-tail (CsRCI2A/B/E/H) type of C-terminal. However, it is still less understood the function of C-terminal tail. In this study, CsRCI2D/H genes were cloned through gateway cloning system that used pCB302-3 as destination vector. And we used agrobacterium-mediated transformation system for generation of overexpression (OX) transformants. Overexpression of target gene was confirmed using RT-PCR and segregation ratio on selection media. We analyzed physiological response in media and soil under abiotic stresses using CsRCI2D and CsRCI2H overexpression plant. To compare abiotic stresses tolerance, wild type and CsRCI2D/H OX line seeds were sown on agar plate treated with various NaCl and mannitol concentration for 7 days. In the test of growth rate under abiotic stress on media, CsRCI2H OX line showed similar to NaCl and mannitol stress. In the other hand, CsRCI2D OX line showed to be improved stress tolerance that especially increased in 200mM NaCl but was similar on mannitol media. In greenhouse, WT and CsRCI2D/H OX lines for physiological analysis and productivity under abiotic stresses were treated 100, 150, 200mM NaCl. Then it was measured various parameters such as leaf width and length, plant height, total seed weight, flower number, seed number. CsRCI2H OX line in greenhouse did not show any changes in physiological parameters but CsRCI2D OX line was improved both physiological response and productivity under NaCl stress. Among physiological parameters of CsRCI2D OX line under NaCl stress, leaf length and width were observed shorter than WT but it were slightly longer than WT in 200mM NaCl stress. Furthermore, total seed weight of CsRCI2D OX line under stress displayed to decrease than WT in normal condition, but it was gradually raised with increasing NaCl stress then more than WT relatively. These results suggested CsRCI2D might be contribute to improve abiotic stress tolerance. However, function of CsRCI2H is need to more detail study. In conclusion, overexpression of CsRCI2s family can generate various environmental stress tolerance plant and may improve crop productivity for bio-energy production.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.8
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• pp.3495-3501
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• 2014

Melanoma-associated antigen (MAGE) family genes have been considered as potentially promising targets for anticancer immunotherapy. MAGED4 was originally identified as a glioma-specific antigen. Current knowledge about MAGED4 expression in glioma is only based on mRNA analysis and MAGED4 protein expression has not been elucidated. In the present study, we investigated this point and found that MAGED4 mRNA and protein were absent or very lowly expressed in various normal tissues and glioma cell line SHG44, but overexpressed in glioma cell lines A172,U251,U87-MG as well as glioma tissues, with significant heterogeneity. Furthermore, MAGED4 protein expression was positively correlated with the glioma type and grade. We also found that the expression of MAGED4 inversely correlated with the overall methylation status of the MAGED4 promoter CpG island. Furthermore, when SHG44 and A172 with higher methylation were treated with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) reactivation of MAGED4 mRNA was mediated by significant demethylation in SHG44 instead of A172. However, 5-AZA-CdR treatment had no effect on MAGED4 protein in both SHG44 and A172 cells. In conclusion, MAGED4 is frequently and highly expressed in glioma and is partly regulated by DNA methylation. The results suggest that MAGED4 might be a promising target for glioma immunotherapy combined with 5-AZA-CdR to enhance its expression and eliminate intratumor heterogeneity.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.11
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• pp.1824-1836
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• 2020

Objective: On the hypothesis that grazing of cattle prompts organs to secrete or internalize circulating microRNAs (c-miRNAs) in parallel with changes in energy metabolism, we aimed to clarify biological events in adipose, skeletal muscle, and liver tissues in grazing Japanese Shorthorn (JSH) steers by a transcriptomic approach. Methods: The subcutaneous fat (SCF), biceps femoris muscle (BFM), and liver in JSH steers after three months of grazing or housing were analyzed using microarray and quantitative polymerase chain reaction (qPCR), followed by gene ontology (GO) and functional annotation analyses. Results: The results of transcriptomics indicated that SCF was highly responsive to grazing compared to BFM and liver tissues. The 'Exosome', 'Carbohydrate metabolism' and 'Lipid metabolism' were extracted as the relevant GO terms in SCF and BFM, and/or liver from the >1.5-fold-altered mRNAs in grazing steers. The qPCR analyses showed a trend of upregulated gene expression related to exosome secretion and internalization (charged multivesicular body protein 4A, vacuolar protein sorting-associated protein 4B, vesicle associated membrane protein 7, caveolin 1) in the BFM and SCF, as well as upregulation of lipolysis-associated mRNAs (carnitine palmitoyltransferase 1A, hormone-sensitive lipase, perilipin 1, adipose triglyceride lipase, fatty acid binding protein 4) and most of the microRNAs (miRNAs) in SCF. Moreover, gene expression related to fatty acid uptake and inter-organ signaling (solute carrier family 27 member 4 and angiopoietin-like 4) was upregulated in BFM, suggesting activation of SCF-BFM organ crosstalk for energy metabolism. Meanwhile, expression of plasma exosomal miR-16a, miR-19b, miR-21-5p, and miR-142-5p was reduced. According to bioinformatic analyses, the c-miRNA target genes are associated with the terms 'Endosome', 'Caveola', 'Endocytosis', 'Carbohydrate metabolism', and with pathways related to environmental information processing and the endocrine system. Conclusion: Exosome and fatty acid metabolism-related gene expression was altered in SCF of grazing cattle, which could be regulated by miRNA such as miR-142-5p. These changes occurred coordinately in both the SCF and BFM, suggesting involvement of exosome in the SCF-BFM organ crosstalk to modulate energy metabolism.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.316-327
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• 2007

In this study, a Korean native cattle strain (Hanwoo) evidencing high performance in terms of both meat quality and quantity was employed in the generation of 150,000 BAC clones with an average insert size of 140 kb, and corresponding to about a 6X coverage of bovine chromosomal DNA. The BAC clones were pooled in a mini-scale via three rounds of a pooling protocol, and the efficiency of this pooling protocol was evaluated by testing the accuracy of accessibility to the positive clones, via a PCR-based screening method. Two sets of primers designed from each of two known genes were tested, and each yielded 2 or 3 positive clones for each gene, thereby indicating that the BAC library pooling system was appropriate with regard to the accession of the target BAC clones. Analyses of $3.3{\times}10^6$ base pairs obtained from the 7,090 BAC end sequence (BES) showed that 34.88% of the DNA sequence harbored the repetition sequence. Analysis of the 7,090 BES to the $1^{st}$ and $2^{nd}$ generation radiation hybrid map of the cattle genome, using the COMPASS program designed for the construction of a cattle-human comparative mapping, resulted in the localization of a total of 1,374 clones proximal to 339 $1^{st}$ generation markers, and 1,721 clones proximal to 664 $2^{nd}$ generation markers. Collectively, the BAC library and pooling system of the BAC clones from the Korean cattle, coupled with the chromosome-localized BAC clones, will provide us with novel tools for the excavation of desired clones for genome mapping and sequencing, and will also furnish us with additional information regarding breed differences in cattle.

• BMB Reports
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• v.53 no.4
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• pp.218-222
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• 2020

Excessive and hyperactive osteoclast activity causes bone diseases such as osteoporosis and periodontitis. Thus, the regulation of osteoclast differentiation has clinical implications. We recently reported that dehydrocostus lactone (DL) inhibits osteoclast differentiation by regulating a nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), but the underlying mechanism remains to be elucidated. Here we demonstrated that DL inhibits NFATc1 by regulating nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and nuclear factor-erythroid 2-related factor 2 (Nrf2). DL attenuated IκBα phosphorylation and p65 nuclear translocation as well as decreased the expression of NF-κB target genes and c-Fos. It also inhibited c-Jun N-terminal kinase (JNK) but not p38 or extracellular signal-regulated kinase. The reporter assay revealed that DL inhibits NF-κB and AP-1 activation. In addition, DL reduced reactive oxygen species either by scavenging them or by activating Nrf2. The DL inhibition of NFATc1 expression and osteoclast differentiation was less effective in Nrf2-deficient cells. Collectively, these results suggest that DL regulates NFATc1 by inhibiting NF-κB and AP-1 via down-regulation of IκB kinase and JNK as well as by activating Nrf2, and thereby attenuates osteoclast differentiation.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.390-394
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• 2004

The genetically modified glyphosate-tolerant soybean contains the following introduced DNA sequences: the EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene from Agrobacterium sp. strain CP4, the 35S promoter from the cauliflower mosaic virus, and the NOS terminator from Agrobacterium tumefaciens. In the present study, detection of these introduced DNAs was performed by amplification using the polymerase chain reaction (PCR). A multiplex PCR method was also applied to prevent false positive results. When primers for 35S promoter, nos3', CTP(chloroplast transit peptide), and CP4 EPSPS (EPSPS from Agrobacterium sp. CP4) were used, positive results were obtained in PCR reactions using DNA from genetically modified glyphosate-tolerant soybeans. There were no false positive results when using DNA from non-genetically modified soybeans. The CP4 EPSPS gene was detected when less than 125 pg glyphosate-tolerant soybean DNA was amplified. Lectin Lel and psb A were amplified from both non-genetically modified and genetically modified glyphosate-tolerant soybean DNA. Multiplex PCR was performed using different primer sets for actin Sacl, 35S promoter and CP4 EPSPS. The actin gene was detectable in both non-genetically modified and glyphosate-tolerant soybeans as a constant endogenous gene. Target DNAs for the 35S promoter, and CP4 EPSPS were detected in samples containing 0.01-0.1% glyphosate-tolerant soybean, although there were variations depending on primers by multiplex PCR. Soybean seeds from five plants of non-genetically modified soybean were co-cultivated for six months with those of genetically modified soybean, and they were analyzed by PCR. As a result, they were not positive for 35S promoter, nos3' or CP4 EPSPS. Therefore, these results suggest there was no natural crossing of genes between glyphosate-tolerant and non-genetically modified soybean during co-cultivation, which indicates that gene transfer between these plants is unlikely to occur in nature.

• Journal of Life Science
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• v.24 no.12
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• pp.1269-1275
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• 2014

TALEN is a newly developed gene engineering method to knock out specific genes. It contains a DNA binding domain and a Fok1 nuclease domain in the TALEN plasmid. Therefore, the engineered TALEN construct can bind to any region of genomic DNA and cut the target nucleotide, thereby inducing mutation. In this study, we constructed two TALEN constructs targeted to a protein initiation codon (DBEX2) or the 25th upstream region (DBPR25) to enable mRNA synthesis of SETDB1 HMTase. We performed the TALEN cloning in two steps. The first step was from module vectors to pFUS array vectors. We confirmed successful cloning with a colony PCR experiment and Esp31 restriction enzyme digestion, which resulted in a smear band and a 1 Kb insert band, respectively The second step of the cloning was from a pFUS array vector to a mammalian TALEN expression vector. The engineered TALEN construct was sequenced with specific primers in an expression vector. As expected, a specific array from the module vectors was shown in the sequencing analysis. The specific module sequences were regularly arrayed in every 100 bp, and SETDB1 expression totally disappeared in the TALEN-DBEX2 transfection. PCR amplification targeting of DBEX2 was performed, and the PCR product was digested with a T7E1 restriction enzyme. The expression of SETDB1 was down-regulated in the TALEN-DBPR25 transfection. Morphological changes were also observed in the two TALEN constructs with transfected HeLa cells. These results suggest that the engineered TALEN constructs in two strategic approaches are very useful to knock-out of the SETDB1 gene and to study gene function.

• Molecules and Cells
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• v.25 no.4
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• pp.559-565
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• 2008

Members of the TGA family of basic domain/leucine zipper transcription factors regulate defense genes through physical interaction with NON-EXPRESSOR OF PR1 (NPR1). Of the seven TGA family members, TGA4/octopine synthase (ocs)-element-binding factor 4 (OBF4) is the least understood. Here we present evidence for a novel function of OBF4 as a regulator of flowering. We identified CONSTANS (CO), a positive regulator of floral induction, as an OBF4-interacting protein, in a yeast two-hybrid library screen. OBF4 interacts with the B-box region of CO. The abundance of OBF4 mRNA cycles with a 24 h rhythm under both long-day (LD) and short-day (SD) conditions, with significantly higher levels during the night than during the day. Electrophoretic mobility shift assays revealed that OBF4 binds to the promoter of the FLOWERING LOCUS T (FT) gene, a direct target of CO. We also found that, like CO and FT, an OBF4:GUS construct was prominently expressed in the vascular tissues of leaf, indicating that OBF4 can regulate FT expression through the formation of a protein complex with CO. Taken together, our results suggest that OBF4 may act as a link between defense responses and flowering.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.2
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• pp.197-204
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• 2018

Multiple myeloma (MM) is the leading cause of death among hematologic neoplasms. Recently, microRNA has been reported to be useful in the diagnosis of multiple myeloma. This study examined whether miR-221 could be used as a diagnostic marker for multiple myeloma. The study was performed on 20 patients with multiple myeloma without any other hematological diseases. MicroRNA extraction was performed using formalin-fixed paraffin-embedded (FFPE) tissues obtained from the bone marrow of patients with multiple myeloma. miR-15a, miR-16, miR-21, miR-181a, and miR-221 were selected as the microRNA target genes for multiple myeloma. The significance of microRNA was based on a fold change of <1.5. To quantify the fold changes, data normalized to the human gene, SNORD43, were used as the values of the patient group. Fold change values greater than 1.5 were defined as "overexpression", whereas values less than -1.5 were defined as "underexpression". Of note, 65.0% (13/20) of samples showed significant "overexpression" in the levels of miR-221 expression and plasma cells with a group of more and less than 30% in MM patients did not show any significance of plasma cell (P<0.05). The results of other studies showing a correlation between the expression of miR-221 and MM in Caucasians were confirmed. These results suggest that miR-221 may be a useful indicator for diagnosing patients with MM. In conclusion, miR-221 is useful in the diagnosis and determining the prognosis of multiple myeloma in Koreans.

• Journal of Life Science
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• v.21 no.7
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• pp.932-938
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• 2011

Ginsenoside Rg1 is a pharmacologically active component isolated from ginseng. The goal of this study was to clarify the beneficial effects of Rg1 on glucose and lipid metabolism in diabetic animals (db/db mice). To accomplish this, ten week old db/db mice were administered 10 mg/kg of Rg1 for 15 days. Rg1 did not influence the weight of db/db mice when compared with vehicle-treated db/db mice. The administration of Rg1 lowered fasting plasma glucose, and improved glucose tolerance. Importantly, Rg1 markedly reduced both plasma triglyceride and free fatty acids, and increased high-density lipoprotein cholesterol (HDL-C) concentrations in db/db mice. Rg1 activated promoter activity of chimeric GAL4-PPAR${\alpha}$ reporter and increased expression of peroxisome proliferator-activated receptor alpha (PPAR${\alpha}$) target genes such as carnitine palmitoyltransferase-1 (CPT-1) and acyl-CoA oxidase (ACO), which are involved in fatty acid oxidation. These findings indicated that improvement of lipid profiles by Rg1 may be associated with increased fatty acid oxidation via PPAR${\alpha}$ activation. Taken together, these results suggest that Rg1 could have beneficial effects for controlling hyperglycemia and hyperlipidemia associated with type 2 diabetes.