• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.74.1-74
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• 2003

Differential display (DD) of mRNA is a technique in which mRNA species expressed by a cell population are reverse transcribed and then amplified by many separate polymerase chain reactions (PCR). Using DD-RT-PCR we obtained many genes that expressed differentially in healthy and PVX-infected Nicotiana benthamima, using total RNAs extracted from healthy and PVX-infected N. benthamiana plants. Three hundred and twenty-five DNA fragments isolated from DD-RT-PCR were cloned and sequenced for further characterization. Several host genes including SKPI-like protein, heat shock transcription factor and Avr9/Cf-9 rapidly elicited protein were selected to obtain full-length open reading frame and to characterize their potential involvement in virus disease development and/or host's defense against virus infection employing PVX-based expression vector. Transcrips from wild-type and clones containing each selected gene were inoculated onto N. benthamiana Levels of virus replication were confirmedby RT-PCR and RNA blot analysis, Expression profiles and potential role(s) of selected genes upon PVX infection will be discussed.

• BMB Reports
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• 제36권1호
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• pp.12-19
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• 2003

Fragmentation of purine imidazole ring and production of formamidopyrimidines in deoxynucleosides (Fapy lesions) occurs upon DNA oxidation as well as upon spontaneous or alkali-triggered rearrangement of certain alkylated bases. Many chemotherapeutic agents such as cyclophosphamide or thiotepa produce such lesions in DNA. Unsubstituted FapyA and FapyG, formed upon DNA oxidation cause moderate inhibition of DNA synthesis, which is DNA polymerase and sequence dependent. Fapy-7MeG, a methylated counterpart of FapyG-, a efficiently inhibits DNA replication in vitro and in E.coli, however its mutagenic potency is low. This is probably due to preferential incorporation of cytosine opposite Fapy-7MeG and preferential extension of Fapy-7MeG:C pair. In contrast, FapyA and Fapy-7MeA possess miscoding potential. Both lesions in SOS induced E.coli preferentially mispair with cytosine giving rise to A$\rightarrow$G transitions. Fapy lesions substituted with longer chain alkyl groups also show simult aneous lethal and mutagenic properties. Fapy lesions are actively eliminated from DNA by repair glycosylases specific for oxidized purines and pyrimidines both in bacteria and eukaryotic cells. Bacterial enzymes include E.coli formamidopyrimidine-DNA-glycosylase (Fpg protein), endonuclease III (Nth protein) and endonuclease VIII (Nei protein).

• The Plant Pathology Journal
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• 제22권2호
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• pp.152-154
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• 2006

CSVd-infected chrysanthemum plants grown under $10^{\circ}C\;or\;15^{\circ}C$ growth chamber for 2 months resulted a higher dilution endpoint of template RNA for Reverse transcription and polymerase chain reaction (RT-PCR) than those grown for 1 month: $10^{-4}(1.35{\times}10^{-2}ug/ml)$ for 1 month, and $10^{-3}(1.35{\times}10^{-1}ug/ml)$ for 2 months. Independent experiment, shoots cut from CSVd (Chrysanthemum stunt viroid)-in footed chrysanthemum plants grown under $10^{\circ}C\;or\;20^{\circ}C$ growth chamber for 2 months showed the same CSVd concentration as control($30^{\circ}C$) at 8 weeks after moving them to normal green-house condition($30^{\circ}C$). From those results, it was concluded that even though the concentration of CSVd was reduced in plants grown at low temperatures, when they were moved to normal glass-house temperature CSVd concentration increased to that of untreated plants in 8 weeks. This conclusion was supported by the rapid replication of CSVd in chrysanthemum after infection.

• Annals of Clinical Neurophysiology
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• 제19권1호
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• pp.40-45
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• 2017

Background: Telbivudine is a nucleoside analogue used for the treatment of chronic hepatitis B, but it often develops mitochondrial toxicity leading to symptomatic myopathy. In this study, three patients with telbivudine induced myopathy were enrolled in order to investigate the nature and pathogenesis of mitochondrial toxicity caused by long-term use of telbivudine. Methods: Clinical features, laboratory findings, muscle pathology, and quantitation of mitochondrial DNA were studied in three patients. Results: Patients presented with progressive muscle weakness with high serum creatine kinase levels. Light microscopic findings of muscle pathology showed ragged red fibers that reacted strongly with succinate dehydrogenase stain, but negative for cytochrome c oxidase activities. Electron microscopy revealed abnormal mitochondrial accumulation with rod shaped inclusions. The quantitative peroxidase chain reaction showed a depletion of mitochondrial DNA in skeletal muscle of the patients. Conclusions: Nucleoside analogues including telbivudine are potent inhibitors of viral DNA polymerases. However, they are not specific for viral DNA and can disturb mitochondrial replication at the same time. All nucleotide analogues should be used with close clinical observation in order to avoid development of mitochondrial myopathy.

• 생명과학회지
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• 제17권10호
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• pp.1361-1367
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• 2007

Nocardiopis sp. 유래의 protein kinase 저해제인 KT5720, KT5926을 사용하여 Vesicular Stomatitis Virus (VSV) 증식에 미치는 세포변성의 억제를 지표로서 연구하였다. 그 결과 CAMP의존 kinase를 저해하는 KT5720, mysion light chain kinase (MLCK)를 저해하는 KT5926이 VSV에 의한 세포변성을 억제시켰다. Protein kinase inhibitor KT5720은 VSV의 증식에 영향을 미치지는 않지만, 세포변성과정은 억제하는 것으로 나타났다. Virus RNA 및 단백질 합성에서 PKA KT5720이 영향을 미치지 않는 것은 virus 증식에 영향이 없는 것으로 나타난다. Virus 증식과정에서 성질이 다른 protein kinase가 관여하는데, 이것은 negative strand virus에 대한 항virus 제 개발에 중요한 역할을 한다고 생각한다.

• International Journal of Oral Biology
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• 제44권2호
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• pp.62-70
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• 2019

Xylitol is well-known to have an anti-caries effect by inhibiting the replication of cariogenic bacteria. In addition, xylitol enhances saliva secretion. However, the precise molecular mechanism of xylitol on saliva secretion is yet to be elucidated. Thus, in this study, we aimed to investigate the stimulatory effect of xylitol on saliva secretion and to further evaluate the involvement of xylitol in muscarinic type 3 receptor (M3R) signaling. For determining these effects, we measured the saliva flow rate following xylitol treatment in healthy individuals and patients with dry mouth. We further tested the effects of xylitol on M3R signaling in human salivary gland (HSG) cells using real-time quantitative reverse-transcriptase polymerase chain reaction, immunoblotting, and immunostaining. Xylitol candy significantly increased the salivary flow rate and intracellular calcium release in HSG cells via the M3R signaling pathway. In addition, the expressions of M3R and aquaporin 5 were induced by xylitol treatment. Lastly, we investigated the distribution of M3R and aquaporin 5 in HSG cells. Xylitol was found to activate M3R, thereby inducing increases in $Ca^{2+}$ concentration. Stimulation of the muscarinic receptor induced by xylitol activated the internalization of M3R and subsequent trafficking of aquaporin 5. Taken together, these findings suggest a molecular mechanism for secretory effects of xylitol on salivary epithelial cells.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.440-448
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• 2021

Helicobacter pylori (H. pylori) establishes long-term infections associated with severe gastric diseases such as peptic ulceration and gastric cancer. Exposure to an antibacterial agent can help regulate the expression levels of its pathogenic genes. In this study, we analyzed the transcriptional changes in H. pylori genes induced by β-caryophyllene. We used next-generation sequencing (NGS) to analyze RNA expression changes, and reverse transcription-polymerase chain reaction (RT-PCR) was performed as required to verify the results. The NGS results showed that 30 out of 1,632 genes were expressed differentially by β-caryophyllene treatment. Eleven genes associated with DNA replication, virulence factors, and T4SS components were significantly downregulated. RT-PCR confirmed that treatment reduced the expression levels of 11 genes. RT-PCR showed the reduced expression of 11 genes (dnaE, dnaN, holB, gyrA, cagA, vacA, secA, flgE, virB2, virB4, and virB8) following β-caryophyllene treatment. These results suggest that β-caryophyllene can modulate various H. pylori pathogenic determinants and be a potential therapeutic agent for H. pylori infection.

• IMMUNE NETWORK
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• 제4권1호
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• pp.16-22
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• 2004

Background: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. Methods: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. Results: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of $4.46{\times}10^9cfu$ was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of $4.5{\times}10^{-7}M$ and $1.9{\times}10^{-7}M$, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. Conclusion: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

• The Plant Pathology Journal
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• 제30권1호
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• pp.58-67
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• 2014

The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic virus (AltMV) has been reported to have silencing suppressor, cell-to-cell movement, and helicase functions. Yeast two hybrid screening using an Arabidopsis thaliana cDNA library with TGB1 as bait, and co-purification with TGB1 inclusion bodies identified several host proteins which interact with AltMV TGB1. Host protein interactions with TGB1 were confirmed by biomolecular fluorescence complementation, which showed positive TGB1 interaction with mitochondrial ATP synthase delta' chain subunit (ATP synthase delta'), light harvesting chlorophyll-protein complex I subunit A4 (LHCA4), chlorophyll a/b binding protein 1 (LHB1B2), chloroplast-localized IscA-like protein (ATCPISCA), and chloroplast ${\beta}$-ATPase. However, chloroplast ${\beta}$-ATPase interacts only with $TGB1_{L88}$, and not with weak silencing suppressor $TGB1_{L88}$. This selective interaction indicates that chloroplast ${\beta}$-ATPase is not required for AltMV movement and replication; however, TRV silencing of chloroplast ${\beta}$-ATPase in Nicotiana benthamiana induced severe tissue necrosis when plants were infected by AltMV $TGB1_{L88}$ but not AltMV $TGB1_{L88}$, suggesting that ${\beta}$-ATPase selectively responded to $TGB1_{L88}$ to induce defense responses.

• Journal of Nutrition and Health
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• 제54권4호
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• pp.335-341
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• 2021

Purpose: Muscle mitochondria play a key role in regulating fatty acid and glucose metabolism. Dysfunction of muscle mitochondria is associated with metabolic diseases such as obesity and type 2 diabetes. Isorhamnetin (ISOR), also known as 3-O-methylquercetin, a quercetin metabolite, is a naturally occurring flavonoid in many plants. This study evaluated the effects of ISOR on the regulation of the mitochondrial function of C2C12 muscle cells. Methods: C2C12 muscle cells were differentiated for 5 days, and then treated in various concentrations of ISOR. Cytotoxicity was determined by assessing cell viability using the water-soluble tetrazolium salt-8 assay principle at different concentrations of ISOR and time points. Levels of the mitochondrial DNA (mtDNA) content and gene expression were measured by quantitative real-time polymerase chain reaction. The citrate synthase (CS) activity was quantified by the enzymatic method. Results: ISOR at a concentration of 10 µM did not show any cytotoxic effects. ISOR increased the mtDNA copy number in a time- or dose-dependent manner. The messenger RNA levels of genes involved in mitochondrial function, such as peroxisome proliferator-activated receptor-γ coactivator-1α, and uncoupling protein 3 were significantly stimulated by the ISOR treatment. The CS activity was also significantly increased in a time- or dose-dependent manner. Conclusion: These results suggest that ISOR enhances the regulation of mitochondrial function, which was at least partially mediated via the stimulation of the mtDNA replication, mitochondrial gene expression, and CS activity in C2C12 muscle cells. Therefore, ISOR may be useful as a potential food ingredient to prevent metabolic diseases-associated muscle mitochondrial dysfunction.