• Research in Plant Disease
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• v.23 no.4
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• pp.363-366
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• 2017

Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV) and Barley yellow dwarf virus (BYDV) have been identified as an important causative agents for an economically important disease of winter barley in Korea. In this study, a multiplex reverse transcription polymerase chain reaction (mRT-PCR) method was used for the simultaneous detection. Three sets of virus-specific primers targeted to the capsid protein coding genes of BaMMV, BaYMV and BYDV were used to amplify fragments that were 594 bp, 461 bp, and 290 bp, respectively. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by multiplex RT-PCR. The optimum primer concentrations and RT-PCR conditions were determined for the multiplex RT-PCR. The mRT-PCR assay was found to be a better and rapid virus diagnostic tool of specific barley diseases and potential for investigating the epidemiology of these viral diseases.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.168-176
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• 2005

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods, namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V vulnificus virulence. The presence of DMSO ($5\%$) and BSA ($0.1\%$) in PCR reaction mixture improved a detection efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around $10^2\;cells\;g^{-1}$ of sample. However, those three methods using the enrichment at $35^{\circ}C$ in APW showed high sensitivity ($2\~10\;cells\;g^{-1}$ of sediments). Highly sensitive detection of V vulnificus by real-time PCR was achieved within $5\~6$ hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V vulnificus in tidal flat sediments.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.4
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• pp.314-320
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• 2001

Matrix metalloproteinases have long been viewed as ideal candidates for proteinases that enables tumor cells to permeated basement membrane defenses and invade surrounding tissue. There is growing evidence that the MMPs have an expanded role, as they are important for the creation and maintenance of a microenvironment that facilitates growth and angiogenesis of tumors at primary and metastatic sites. MT-MMPs are not secreted but instead remaining attached to cell surfaces. Although not all of the MT-MMPs are fully characterized, MT-MMPs have important role in localizing and activating secreted MMPs. The MMP genes are transcriptionally responsive to a wide variety of oncogene, growth factors, cytokine, and hormones. Currently, a number of MMP inhibitors are being developed and some have reached clinical trials as anti-metastatic or anti-cancer therapies. MT1-MMP is involved in the activation of proMMP-2. MT1-MMP is significant not only as a tumor marker but as a new target for chemotherapy against cancer. The purpose of this study was to evaluate the effects of protein kinase C inhibitor(genistein) on the proliferation of HT1080 and expression of MT1-MMP mRNA. Human fibrosarcoma cell line HT1080 was cultured and divided 2 groups. The experimental group was treated with $100{\mu}M$ genistein and incubated 12h, 24h for $[3^H]-thymidine$ uptake assay and northern hybridization individually. And the control group was treated with same amount of PBS for the above procedures. $[3^H]-thymidine$ incorporation was measured with ${\beta}$ ray detector. And RT-PCR and northern blotting for MT1-MMP mRNA was performed. The results were as follows 1. $[3^H]-thymidine$ uptake was reduced in experimental group with statistical significance. 2. MT1-MMP mRNA expression was significantly reduced in experimental group. These results showed that protein kinase C inhibitor (genistein) inhibited proliferation of HT1080 and almost completely blocked transcription of MT1-MMP mRNA. So, it is possible to use the protein kinase inhibitor (genistein) as anti-metastatic and anti-proliferative agent.

• Korean Journal of Pharmacognosy
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• v.48 no.3
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• pp.179-186
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• 2017

Pyrrosia lingua which belongs to Polypodiaceae has been used as a traditional medicine for the treatment of urinary system inflammation, urination disorder, and bronchitis. However, there are not enough phytochemical and pharmacological studies of P. lingua up to now. Here in this study, the protective effect of MeOH extract of whole plant of Pyrrosia lingua (MPL) against 2% glucose-induced toxicity was investigated using Caenorhabditis elegans (C. elegans) model system. We found that MPL significantly extended the lifespan of wild-type nematode under normal culture condition. MPL also effectively recovered the decreased lifespan caused by 2% glucose-toxicity. In addition, MPL efficiently attenuated the increased glucose concentration inside of nematode. Further studies evaluating diabetes-related factors revealed that MPL reduced both intracellular ROS and lipid accumulation which were up-regulated under 2% glucose supplement condition. Our data also showed that MPL improved the 2% glucose-induced shortened body movement of nematode. Lastly, we carried out genetic studies using several single gene knockout mutants to establish the possible target of MPL. Our results demonstrated that genes such as daf-2 and daf-16 were responsible for the protective activity of MPL against 2% glucose-induced toxicity. These results indicate that MPL exerts protective action against 2% glucose via regulation of insulin/IGF-1 sinaling pathway and FOXO activation.

• The Korean Journal of Pesticide Science
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• v.18 no.4
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• pp.422-428
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• 2014

Endophytic bacterial strains from root tissue of strawberry were screened for their efficacy in growth improvement and control of Phytophthora blight disease of chili pepper plant under greenhouse condition. Plants treated with the strain EP103, identified as Pseudomonas fluorescens, showed growth improvement in terms of fresh weight and root length compared to the untreated control and other endophytic strains. When challenged with Phytophthora capsici, there was significant reduction of disease in EP103 treated plants with an efficacy of 78.7%. There was no direct inhibition of the target pathogen by EP103 when tested under in vitro antibiosis assay. Analysis of differential expression of selected marker genes for induced systemic resistance (ISR) in plants treated with EP103 and challenged with P. capsici showed up-regulation of PR1 and PR10 pathogenesis-related (PR) proteins. PCR analysis showed that EP103 produced secondary metabolites such as pyoluteorin, pyrrolnitrin, hydrogen cyanide and orfamide A. This study indicated the potential of endophytic P. fluorescens strain EP103 as an efficient biocontrol agent against P. capsici in chili pepper plant.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.22
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• pp.9973-9978
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• 2014

Background: Hypoxia inducible factor-1 alpha (HIF-$1{\alpha}$) is the key regulator of cellular responses to hypoxia and plays a central role in tumour growth. Presence of Single nucleotide polymorphisms (SNPs) in the critical regulatory domains of HIF-$1{\alpha}$ may result in the overexpression of the protein and subsequent changes in the expression of the downstream target genes. The aim of study was to investigate the association of three SNPs (g.C111A, g.C1772T and g.G1790A) of HIF-$1{\alpha}$ with the risk of breast cancer in North Indian sporadic breast cancer patients. Materials and Methods: A total of 400 subjects, including 200 healthy controls and 200 patients with breast cancer were recruited in this study. Genotypes were determined using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method. Results: The CC and CA genotype frequency of HIF-$1{\alpha}$ g.C111A polymorphism was 100 vs 99% and 0 vs 1% in breast cancer patients and healthy controls respectively. The frequencies of CC, CT and TT genotype of g.C1772T polymorphism were 76 vs 74.5%, 19 vs 21% and 5 vs 4.5% in breast cancer patients and control individuals respectively. There was no significant difference in genotype and allele frequencies of HIF-$1{\alpha}$ g.C1772T polymorphism between cases and control individuals (p>0.05). For g.G1790A genotypes, all patients and controls had only GG genotype. Conclusions: The three HIF-$1{\alpha}$ polymorphisms (g.C111A, g.C1772T and g.G1790A) are not associated with breast cancer risk in North-West Indian patients.

• Proceedings of the SCSK Conference
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• 2003.09a
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• pp.780-803
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• 2003

Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB ($\textrm{NF}_{-{\kappa}}\textrm{B}$) activators [e.g. tumor necrosis factor-$\alpha$, interleukin-1, lipopolysaccharides, and ultraviolet light] leads to phosphorylation and degradation of the inhibitory protein, $\textrm{I}_{{\kappa}}\textrm{B}$. Liberated $\textrm{NF}_{-{\kappa}}\textrm{B}$ is translocated into the nucleus where it can change or alter expression of target genes, resulting in the secretion of extracellular signaling molecules including melanotrophic factors affecting melanocyte. In order to demonstrate the possible role of $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of $\textrm{NF}_{-{\kappa}}\textrm{B}$ induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with $\textrm{pNF}_{-{\kappa}}\textrm{B}$-SEAP-NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the $\textrm{NF}_{-{\kappa}}\textrm{B}$ activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selection marker for geneticin resistance. MIs such as niacinamide, kojic acid, hydroquinone, resorcinol, arbutin, and glycolic acid were preincubated with transfectant HaCaT cells for 3 h and then ultraviolet B (UVB) was irradiated. $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation was measured with the SEAP reporter gene assay using a fluorescence detection method. Of the Mis tested, kojic acid ($IC_{50}$/ = 60 $\mu$M) was found to be the most potent inhibitor of UVB-upregulating $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation in transfectant HaCaT cells, which is followed by niacinamide ($IC_{50}$/= 540 $\mu$M). Pretreatment of the transfectant HaCaT cells with the Mis, especially kojic acid and niacinamide, effectively lowered $\textrm{NF}_{-{\kappa}}\textrm{B}$ binding measured by electrophoretic mobility shift assay. Furthermore, these two inhibitors remarkably reduced the secretion level of IL-6, one of melanotrophic factors, triggered by UV-radiation of the HaCaT cells. These observations suggest that Mis working at the in vivo level might act partially through the modulation of the synthesis of melanotrophic factors in keratinocyte.

• The Korea Journal of Herbology
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• v.30 no.4
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• pp.57-64
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• 2015

Objectives : Oxidative stress is one of the most causes of hepatocyte injury. Gleditsia spina, the thorns ofGleditsia sinensisLam., has been known for its anti-cancer and anti-inflammatory effects in Korean medicine. The present study investigated hepatoprotective effect of Gleditsia spina water extract (GSE) against oxidative stress induced by arachidonic acid (AA) + iron in HepG2 cells.Methods : To investigate cytoprotective effect of GSE, cells were pretreated with GSE and then subsequently exposed to 10 μM AA for 12 h, followed by 5 μM iron. Cell viability was monitored by MTT assay, and expression of apoptosis-related proteins was examined by immunoblot analysis. To identify responsible molecular mechanisms, reactive oxygen species (ROS) production, GSH contents, and mitochondrial membrane potential were measured. In addition, effect of GSE on nuclear factor erythroid 2-related factor 2 (Nrf2) activation was determined by immunoblot and antioxidant response element (ARE)-driven reporter gene assays.Results : GSE pretreatment prevented AA + iron-mediated cytotoxicity in concentration dependent manner. In addition, ROS production, glutathione depletion, and mitochondrial impairment by AA + iron were significantly inhibited by GSE. Furthermore, GSE promoted translocation of Nrf2 to nucleus, which acts as essential transcription factor for induction of antioxidant genes. Increased nuclear Nrf2 that caused by GSE treatment promoted transcriptional activity of ARE. Finally, GSE up-regulated sestrin-2 which was widely recognized as target gene of Nrf2.Conclusions : This study demonstrates that GSE protects hepatocytes from oxidative stress via activation of Nrf2 signaling pathway.

• Journal of Forest and Environmental Science
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• v.30 no.2
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• pp.243-252
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• 2014

Forest tree species usually takes for long periods to be harvested and cultivated but spearmints are a good model system for woody plant because of reducing and shortening cultivation time. Spearmints are good model plants (Mentha species) for research about terpenoids production and industrial essential oil manufacture. Isopentenyl pyrophosphate isomerase (Iso) and limonene synthase (Limo) are the key enzymes of terpenoid biosynthesis pathway. Transgenic and wild spearmints (Mentha spicata, MS) were cultured in vitro and assessed for the essential oil contents. The content of essential oil of transgenic spearmint also was enhanced slightly depending on the target terpenoid genes. In an attempt to increase productivity of terpenoids further, salt and fungal elicitation strategy was adopted on transgenic Mentha spicata. The salt (800 mM NaCl) as abiotic and two fungi (Botrytis cinerea and Glomerella cingulata) as biotic were used for elicitors. In the absence of salt stress four terpenoids were detected from the spearmint extracts, all of them being monoterpenes. On the other hand, the transgenic (MSIso) extracts contained eleven terpenoids (10 monoterpenes and 1 phenylpropene) while transgenic (MSLimo) extracts contained seven monoterpenes. After 3 days of fungal infection, the resistance indices further increased to 4.38, 3.89 and 2.04 for wild type, MSIso and MSLimo, respectively. The salt and fungal elicitators proved beneficial towards modifying both the terpenoids profile and improvement in the composition of essential oil. These results have important applications for the large-scale production of essential oils and forest biotechnology with respect to spearmint.

• Molecules and Cells
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• v.37 no.7
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• pp.547-553
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• 2014

Glioblastoma multiforme (GBM) is one of the most common brain malignancies and has a very poor prognosis. Recent evidence suggests that the presence of cancer stem cells (CSC) in GBM and the rare CSC subpopulation that is resistant to chemotherapy may be responsible for the treatment failure and unfavorable prognosis of GBM. A garlic-derived compound, Z-ajoene, has shown a range of biological activities, including anti-proliferative effects on several cancers. Here, we demonstrated for the first time that Z-ajoene specifically inhibits the growth of the GBM CSC population. CSC sphere-forming inhibition was achieved at a concentration that did not exhibit a cytotoxic effect in regular cell culture conditions. The specificity of this inhibitory effect on the CSC population was confirmed by detecting CSC cell surface marker CD133 expression and biochemical marker ALDH activity. In addition, stem cell-related mRNA profiling and real-time PCR revealed the differential expression of CSC-specific genes, including Notch, Wnt, and Hedgehog, upon treatment with Z-ajoene. A proteomic approach, i.e., reverse-phase protein array (RPPA) and Western blot analysis, showed decreased SMAD4, p-AKT, 14.3.3 and FOXO3A expression. The protein interaction map (http://string-db.org/) of the identified molecules suggested that the AKT, ERK/p38 and $TGF{\beta}$ signaling pathways are key mediators of Z-ajoene's action, which affects the transcriptional network that includes FOXO3A. These biological and bioinformatic analyses collectively demonstrate that Z-ajoene is a potential candidate for the treatment of GBM by specifically targeting GBM CSCs. We also show how this systemic approach strengthens the identification of new therapeutic agents that target CSCs.