• Journal of Ginseng Research
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• v.40 no.2
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• pp.176-184
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• 2016

Background: Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods: The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results: In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion: This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

• The Plant Pathology Journal
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• v.30 no.1
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• pp.33-42
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• 2014

Nivalenol (NIV) and deoxynivalenol (DON) are predominant Fusarium-producing mycotoxins found in grains, which are mainly produced by Fusarium asiaticum and F. graminearum. NIV is found in most of cereals grown in Korea, but the genetic basis for NIV production by F. asiaticum has not been extensively explored. In this study, 12 genes belonging to the trichothecene biosynthetic gene cluster were compared at the transcriptional level between two NIV-producing F. asiaticum and four DON-producing F. graminearum strains. Chemical analysis revealed that time-course toxin production patterns over 14 days did not differ between NIV and DON strains, excluding F. asiaticum R308, which was a low NIV producer. Both quantitative real-time polymerase chain reaction and Northern analysis revealed that the majority of TRI gene transcripts peaked at day 2 in both NIV and DON producers, which is 2 days earlier than trichothecene accumulation in liquid medium. Comparison of the gene expression profiles identified an NIV-specific pattern in two transcription factor-encoding TRI genes (TRI6 and TRI10) and TRI101, which showed two gene expression peaks during both the early and late incubation periods. In addition, the amount of trichothecenes produced by both DON and NIV producers were correlated with the expression levels of TRI genes, regardless of the trichothecene chemotypes. Therefore, the reduced production of NIV by R308 compared to NIV or DON by the other strains may be attributable to the significantly lower expression levels of the TRI genes, which showed early expression patterns.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.4
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• pp.489-498
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• 2018

Objective: Foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV) and PRRS virus (PRRSV), the present study introduced two genetic modification techniques to porcine cells. Methods: First, cluster of differentiation 163 (CD163), the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs) were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7) gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results: shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion: We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

• ALGAE
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• v.35 no.2
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• pp.177-187
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• 2020

Cyanobacteria have been widely reported to produce a variety of UV-absorbing mycosporine-like amino acids (MAAs). Herein, we reported production of the unusual MAA, mycosporine-glycine-alanine (MGA) in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using a newly developed UHPLC-DAD-MS/HRMS (ultra-high performance liquid chromatography-diode array detection-high resolution tandem mass spectrometry) method. MGA had previously been first identified in a red-algae, but S. torques-reginae strain ITEP-024 is the first cyanobacteria to be reported as an MGA producer. Herein, the chemical structure of MGA is fully elucidated from one-dimensional / two-dimensional nuclear magnetic resonance and HRMS data analyses. MAAs are unusually produced constitutively in S. torques-reginae ITEP-024, and this production was further enhanced following UV-irradiance. It has been proposed that MAA biosynthesis proceeds in cyanobacteria from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate. Annotation of a gene cluster encoded in the genome sequence of S. torques-reginae ITEP-024 supports these gene products could catalyse the biosynthesis of MAAs. However, addition of glyphosate to cultures of S. torques-reginae ITEP-024 abolished constitutive and ultra-violet radiation induced production of MGA, shinorine and porphyra-334. This finding supports involvement of the shikimic acid pathway in the biosynthesis of MAAs by this species.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.68-72
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• 2002

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin is present in the two closely related bacterial species, Streptomyces lividans and Streptomyces coelicolor, it normally is expressed only in S. coelicolor---generating the deep blue colonies responsible for the S. coelicolor name. However, multiple copies of the afsR2 gene, which activates actinorhodin synthesis, result in the ability of S. lividansto also synthesize large amounts of actinorhodin. Here we report that the phenotypic property that historicially distinguishes these two Streptomycesspecies is determined conditionally by the carbon source used for culture. Whereas growth on glucose repressed actinorhodin production in S. lividans, culture on solid media containing glycerol as the sole carbon source dramatically increased the expression of afsR2 mRNA---leading to extensive actinorhodin synthesis by S. lividansand obliterating its phenotypic distinction from S. coelicolor. afsR2 transcription under these conditions was developmentally regulated, rising sharply at the time of aerial mycelium formation and coinciding temporally with the onset of actinorhodin production. Our results, which identify media-dependent parallel pathways that regulate actinorhodin synthesis in S. lividans, demonstrate carbon source control of actinorhodin production through the regulation of afsR2 mRNA synthesis. The nucleotide sequences of afsR2 revealed two putative important domains; the domain containing direct repeats in the middle and the domain homologous to sigma factor sequence in the C-terminal end. In this work, we constructed various sized afsR2-derivatives and compared the actinorhodin stimulating effects in S. lividans TK21. The experimental data indicate that the domain homologous to sigma factor sequence in the C-terminal end of afsR2 plays a critical role as an antibiotic stimulating function. In addition, we also observed that the single copy integration of afsR2 regulatory gene into S. lividans TK21 chromosome significantly activates antibiotic overproduction.

• Microbiology and Biotechnology Letters
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• v.43 no.1
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• pp.22-30
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• 2015

CSF is a major concern for the swine industry, representing currently the most epizootically dangerous disease to the species. Numerous CSFV isolates with various degrees of virulence have already been isolated worldwide, ranging from low virulent strains that do not result in any apparent clinical signs to highly virulent strains that cause a severe per acute hemorrhagic fever with very high mortality. The molecular epidemiology of CSFVs has proven to be an essential tool for effective disease control and the development of safe and effective vaccines. Therefore, this study cloned and sequenced local CSFV isolates, and conducted a phylogenetic analysis based on the E2 glycoprotein encoding sequences.The RNA was extracted from PK15 cell culture passaged CSFV isolates, the cDNA prepared, and the complete E2 gene amplified with a product size of 1186 bp. The gelpurified PCR product was cloned into a pGEMT easy vector and the positive clone commercially sequenced. Aligning the nucleotide (1119 bp) and amino acid (373) sequences with 29 reference strains revealed nucleotide and amino acid sequence identities of 82.60-97.80% and 88.70-98.70%, respectively, indicating a higher mutation rate of the field CSFV strains. The phylogenetic analysis based on the complete E2 amino acid sequences also revealed a reliable differentiation of all the analyzed strains into specific genetic groups and subgroups, plus the local isolate (CSFV-E2) was found to cluster with the CSFV subgroup 2.2. Thus, the full-length E2 cds proved to be most suitable for a reliable and statistically significant phylogenetic analysis of CSFV isolates.

• Fisheries and Aquatic Sciences
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• v.20 no.7
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• pp.9.1-9.13
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• 2017

Background: Metal-responsive transcription factor-1 (MTF-1) is a key transcriptional regulator playing crucial roles in metal homeostasis and cellular adaptation to diverse oxidative stresses. In order to understand cellular pathways associated with metal regulation and stress responses in Pacific abalone (Haliotis discus hannai), this study was aimed to isolate the genetic determinant of abalone MTF-1 and to examine its expression characteristics under basal and experimentally stimulated conditions. Results: The abalone MTF-1 shared conserved features in zinc-finger DNA binding domain with its orthologs; however, it represented a non-conservative shape in presumed transactivation domain region with the lack of typical motifs for nuclear export signal (NES) and Cys-cluster. Abalone MTF-1 promoter exhibited various transcription factor binding motifs that would be potentially related with metal regulation, stress responses, and development. The highest messenger RNA (mRNA) expression level of MTF-1 was observed in the testes, and MTF-1 transcripts were detected during the entire period of embryonic and early ontogenic developments. Abalone MTF-1 was found to be Cd inducible and highly modulated by heat shock treatment. Conclusion: Abalone MTF-1 possesses a non-consensus structure of activation domains and represents distinct features for its activation mechanism in response to metal overload and heat stress. The activation mechanism of abalone MTF-1 might include both indirect zinc sensing and direct de novo synthesis of transcripts. Taken together, results from this study could be a useful basis for future researches on stress physiology of this abalone species, particularly with regard to heavy metal detoxification and thermal adaptation.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.109-120
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• 2014

The epothilones are a class of microtubule inhibitors that exhibit a strong antitumor activity. UTD2 is a novel epothilone analog generated by genetic manipulation of the polyketide biosynthetic gene cluster. This study investigated the effects of UTD2 on the actin cytoskeleton and its critical regulators, and the signaling pathways which are essential for cell motility, growth and survival in MCF-7 breast cancer cells. Results showed that UTD2 inhibited the cellular functions of actin cytoskeleton, such as wound-closure, migration and invasion, as well as adhesion. Our study further demonstrated that UTD2 suppressed Rac1 GTPase activation and reduced the activity of PAK1, which is a downstream effector of Rac1, while the activity of Cdc42 was not affected. Additionally, the phosphorylation of p38 and ERK were significantly inhibited, but the phosphorylation of JNK remained the same after UTD2 treatment. Moreover, UTD2 inhibited the activity and mRNA expression of MMP-2, which plays a key role in cell motility. UTD2 also reduced the phosphorylation of Akt, which is an important signaling kinase regulating the cell survival through Rac1. Furthermore, UTD2 interrupted the synergy between Rac1 and Raf in focus formation assays. Taken together, these results indicated that UTD2 exerted multiple effects on the actin cytoskeleton and signaling pathways associated with Rac1. This study provided novel insights into the molecular mechanism of the antineoplastic and antimetastatic activities of epothilones. Our findings also suggest that the signaling pathways regulated by Rac1 may be evaluated as biomarkers for the response to therapy in clinical trials of epothilones.

• Proceedings of the KSRS Conference
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• 2007.10a
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• pp.438-441
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• 2007

Recently, due to molecular biology and engineering technology, DNA microarray makes people watch thousands of genes and the state of variation from the tissue samples of living body. With DNA Microarray, it is possible to construct a genetic group that has similar expression patterns and grasp the progress and variation of gene. This paper practices Cluster Analysis which purposes the discovery of biological subgroup or class by using gene expression information. Hence, the purpose of this paper is to predict a new class which is unknown, open leukaemia data are used for the experiment, and MCL (Markov CLustering) algorithm is applied as an analysis method. The MCL algorithm is based on probability and graph flow theory. MCL simulates random walks on a graph using Markov matrices to determine the transition probabilities among nodes of the graph. If you look at closely to the method, first, MCL algorithm should be applied after getting the distance by using Euclidean distance, then inflation and diagonal factors which are tuning modulus should be tuned, and finally the threshold using the average of each column should be gotten to distinguish one class from another class. Our method has improved the accuracy through using the threshold, namely the average of each column. Our experimental result shows about 70% of accuracy in average compared to the class that is known before. Also, for the comparison evaluation to other algorithm, the proposed method compared to and analyzed SOM (Self-Organizing Map) clustering algorithm which is divided into neural network and hierarchical clustering. The method shows the better result when compared to hierarchical clustering. In further study, it should be studied whether there will be a similar result when the parameter of inflation gotten from our experiment is applied to other gene expression data. We are also trying to make a systematic method to improve the accuracy by regulating the factors mentioned above.

• Molecules and Cells
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• v.40 no.10
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• pp.731-736
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• 2017

Taste sensitivity to sugars plays an essential role in the initiation of feeding behavior. In Drosophila melanogaster, recent studies have identified several gustatory receptor (Gr) genes required for sensing sweet compounds. However, it is as yet undetermined how these GRs function as taste receptors tuned to a wide range of sugars. Among sugars, fructose has been suggested to be detected by a distinct receptor from other sugars. While GR43A has been reported to sense fructose in the brain, it is not expressed in labellar gustatory receptor neurons that show taste response to fructose. In contrast, the Gr64a-Gr64f gene cluster was recently shown to be associated with fructose sensitivity. Here we sought to decipher the genes required for fructose response among Gr64a-Gr64f genes. Unexpectedly, the qPCR analyses for these genes show that labellar expression levels of Gr64d and Gr64e are higher in fructose low-sensitivity flies than in high-sensitivity flies. Moreover, gustatory nerve responses to fructose in labellar sensilla are higher in Gr64d and Gr64f mutant lines than in mutant flies of the other Gr64a-Gr64f genes. These data suggest the possibility that deletion of GR64D or GR64F may indirectly induce enhanced fructose sensitivity in the labellum. Finally, we conclude that response to fructose cannot be explained by a single one of the Gr64a-Gr64f genes.