• Research in Plant Disease
• /
• v.12 no.2
• /
• pp.139-143
• /
• 2006

Virion captured reverse transcription polymerase chain reaction (VC/RT-PCR) could detect plant virus quickly and accurately. In the VC/RT-PCR, no antibody is needed unlike immuno-captured RT-PCR (IC/RT-PCR) which had been improved method of RT-PCR for plant viruses, and virus nucleic acids can be obtained easily within 30minutes by property of polypropylene PCR tube which is hold and immobilized viral particles on its surface. For the virion capture of Tomato spotted wilt virus (TSWV), the extraction buffer was tested. The optimum macerating buffer for TSWV was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. The viral crude sap was incubated for 30 min at $4^{\circ}C$. The virions in the PCR tubes were washed two times with 0.01M PBS containing 0.05% Tween-20. The washed virions were treated at $95^{\circ}C$ immediately for 1 min containing RNase free water and chilled quickly in the ice. Disclosed virions' RNAs by heat treatment were used for RT-PCR. Dilution end point of $10^{-5}$ from plant's crude sap infected with TSWV showed relatively higher detection sensitivity for VC/RT-PCR. During multiple detection using two or more primers, interference was arisen by interactions between primer-primer and plant species. The result of multiplex RT-PCR was influenced by combinations of primers and the kind of plant, and the optimum extraction buffer for the multiplex detection by VC/RT-PCR should be developed.

• Journal of the Korean Applied Science and Technology
• /
• v.36 no.2
• /
• pp.394-406
• /
• 2019

The cellular senescence may be due to damage by the reactive oxygen species (ROS). This study has compared the antioxidant activity in the human cell lines of various origins, including 10S and 50S-derived normal skin fibroblasts, and 10S bone marrow, dental tissue and adipose-derived adult stem cells. After being exposed to $H_2O_2$, half inhibitory concentration ($IC_{50}$) values by cytotoxicity assay was significantly (P<0.05) lower in 50S-derived skin fibroblasts, than in 10S-derived skin fibroblasts and various adult stem cell lines. The cell population doubling time (PDT) and the cell frequency with high senescence associated-${\beta}$-galactose activity were remarkably increased in 50S-derived fibroblasts exposed to 50 ppm $H_2O_2$ for 7 days, than those of 10S-derived fibroblasts and various adult stem cell lines. Further, the expression level of antioxidant-related genes, glutathione peroxidase (GPX) and catalase (CAT), was investigated in 10S and 50S-derived skin fibroblasts, and 10S-derived various adult stem cells by reverse transcription polymerase chain reaction (RT-PCR). The expression level of GPX was higher in most of cell lines, compared to CAT, and a significantly (P<0.05) higher expression level of GPX was observed in 10S-derived skin fibroblasts and adult stem cell lines, compared to 50S-derived skin fibroblasts. We concluded that old-aged skin fibroblasts seemed to be less resistant against ROS than young-aged skin fibroblasts and adult stem cells.

• Journal of Life Science
• /
• v.29 no.3
• /
• pp.303-310
• /
• 2019

The purpose of the study was to compare the effect of either moderate or high intensity aerobic exercise on inflammasome, M1, M2 macrophage infiltration and brown adipocyte markers in subcutaneous adipose tissue of the high fat diet-induced obese mice. The 4 weeks male C57BL/6 mice were randomly assigned to four groups: normal diet control (NC; n=10), high-fat diet control (HC; n=10), high fat diet with moderate intensity exercise (HME; n=10), or high fat diet with high intensity exercise (HIE; n=10) groups. The high fat diet was given 60% calories from fat whereas normal diet was given 18% calories from fat. The moderate intensity exercise group (HME) was set at 10m/min in the first 2 weeks, 12m/min in 3-5 weeks and 14m/min in 6-16 weeks and the high intensity exercise group (HIE) was set at 14m/min in the first 2 weeks, 17m/min in 3-5 weeks and 18m/min in 6-16 weeks. The semi quantitative reverse transcription-polymerase chain reaction (RT PCR) was used to analyze the gene expression. The moderate intensity exercise significantly reduced the expression of NLRP3, F480, CD11c and CD86. Further, the moderate intensity exercise significantly increased CD206 and $PGC1{\alpha}$, BMP7 and PRDM. The high intensity exercise significantly reduced NLRP3, CD11c and CD86. Further, the high intensity exercise significantly increased $PGC1{\alpha}$ and BMP7. In conclusion, moderate intensity exercise has more positive effects on inflammasome, M1, M2 macrophage infiltration and brown adipocyte maskers compared to high intensity exercise in high fat diet induced obese mice.

• Pediatric Infection and Vaccine
• /
• v.26 no.2
• /
• pp.99-111
• /
• 2019

Purpose: Respiratory syncytial virus (RSV) and human rhinovirus (hRV) are the most common causes of child respiratory viral infections. We aimed to investigate epidemiological and clinical characteristics of RSV and hRV single infections and coinfections. Methods: Nasopharyngeal aspirates of hospitalized children aged <5 years were tested using multiplex reverse transcription polymerase chain reaction (RT-PCR) from October 2014 to April 2017. Their medical records were retrospectively reviewed. Results: RSV or hRV was detected in 384 patients who divided into 3 groups: patients with RSV (R group, n=258); patients with hRV (H group, n=99); and patients with both (RH group, n=27). The R group (median age, 6 months) consisted of 248 (96.1%) patients with lower respiratory tract infection (LRTI), and 14 (5.4%) needed oxygen inhalation. Infants aged <12 months (63.2%) had respiratory difficulty and were supplied oxygen more often. The H group (median age, 16 months) consisted of 56 (56.6%) patients with LRTI, 4 (4%) required oxygen inhalation, and 1 (1.0%) required mechanical ventilation. Infants (40.4%) showed longer hospitalization compared to patients aged ${\geq}12$ months (5 vs. 4 days, P<0.05). The RH group consisted of 24 (88.9%) patients with LRTI, and 2 (7.4%) needed oxygen inhalation. Hospitalization days and oxygen inhalation and mechanical ventilation rates did not differ between single infections (R and H groups) and coinfections (RH group). Conclusions: RSV was detected more often in younger patients and showed higher LRTI rates compared to hRV. Single infections and coinfections of RSV and hRV showed no difference in severity.

• Journal of Plant Biotechnology
• /
• v.46 no.3
• /
• pp.228-235
• /
• 2019

Apple (Malus pumila) is one of the most economically important fruits in Korea. but virus infection has decreased the sustainable production of apples and caused serious problems such as yield loss and poor fruit quality. Virus or viroid infection including apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple mosaic virus (ApMV) and apple scar skin viroid (ASSVd) have been also reported in Korea. In many cases, as apple gets infected with virus and viroid with no specific symptoms, the damage and symptoms caused by the viruses are not detected. In our research, viruses in the rootstock were eliminated for a virus-free apple dwarfing rootstock of M.9 and M.26. The virus elimination methods were apical meristem culture, thermotherapy ($37^{\circ}C$, 6 weeks) and chemotherapy($Ribavirin^{(R)}$). The detection of apple viruses was accomplished by Enzyme-linked Immuno-Sorbent Assay (ELlSA) and reverse transcription-polymerase chain reaction (RT-PCR). RT- PCR method was 10 ~ 30% more sensitive than the ELISA method. The efficiency of virus elimination was enhanced in apical meristem culture method. The acquisition rate of virus-free apple dwarfing rootstocks was 30 ~ 40% higher in apical meristem culture. After the meristem culturing of M.9, the infection ratio of ACLSV, ASPV and ASGV was 45%, 60% and 50%, respectively. In the apple dwarfing rootstock of M.26, the infection ratio of ACLSV, ASPV and ASGV was 40%, 55% and 55%, respectively. Based on this study, the best method for the production of virus-free apple dwarfing rootstocks was the apical meristem culture.

• Journal of Life Science
• /
• v.29 no.8
• /
• pp.895-903
• /
• 2019

The aim of the present study was to improve the cell viability of human dental papilla derived single-induced pluripotent stem cells (iPSCs) using a Rho-associated protein kinase (ROCK) inhibitor, Y-27632. The iPSCs were produced using an episomal plasmid-based reprogramming method. After cell separation using trypsin, the iPSCs were treated with 0, 0.5, 1, 2.5, 5, 7.5, or $10{\mu}M$ Y-27632 for 5 d. Cell viability increased significantly following the $5{\mu}M$ Y-27632 treatment (p<0.05). When the iPSCs were exposed to medium containing $10{\mu}M$ Y-27632 for 0, 1, 2, 3, 4, and 5 d, the cell viability rate increased significantly in accordance with the cell viability rate (p<0.05). To evaluate the effect of the Y-27632 treatment on stemness characteristics, the expression of stem cell-specific transcripts and telomerase activity were investigated in the iPSCs treated with $10{\mu}M$ Y-27632 for 5 d. The expression levels of stem cell-specific transcripts, such as OCT-4, NONOG, and SOX-2, and telomerase activity were not significantly different in the iPSCs treated with $10{\mu}M$ Y-27632 as compared with those of untreated control iPSCs (p>0.05). Taken together, the results demonstrated that cell viability can be improved by treatment with the ROCK inhibitor Y-27632, without losing iPSC stemness characteristics.

• Asian-Australasian Journal of Animal Sciences
• /
• v.32 no.7
• /
• pp.922-929
• /
• 2019

Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3'-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3'-UTR and pGL3 Control LDLR mutant-3'-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine premiR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3'-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR3'-UTR-driven (p<0.01), but not mutant LDLR-3'-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = -0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3'-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

• Research in Plant Disease
• /
• v.27 no.1
• /
• pp.1-7
• /
• 2021

In August 2020, necrotic ringspots on leaves were observed on 20 from 143 Plantago asiatica plants in open fields in Eumseong, Chungcheongbuk-do. Eight symptomatic Plantago asiatica plants were subjected to investigation on viral infection by reverse transcription and polymerase chain reaction. Impatiens necrotic spot virus, tomato spotted wilt virus and cucumber mosaic virus were detected from the symptomatic plants. Two impatiens necrotic spot virus (INSV) isolates ('INSV-plantain kr1' and '-plantain kr5') were sequenced and analyzed by comparing L genomic segment, nucleoprotein (N) gene and non-structural protein S (NSs) gene sequences. The nucleotide sequence of 'INSV-plantain kr1' isolate (MW114834) was most closely related to that of a 'Phalaenopsis' isolate (GQ336991) from China in the L genomic segment. 'INSV-plantain kr1' and '-plantain kr5' isolates shared the highest identities with those from 'Pepe' isolate (LC384872) and 'J' isolate (AB109100) in the NSs gene, respectively, and with that from 'YSMi-SH' isolate (FN400773) in the N gene. Phylogenetic analysis based on L genomic segment grouped the INSV-plantain kr1 isolate together with isolates from Korea (LC384870), China (GU112505, GQ336991), and Italy (DQ425094). This is the first report on INSV in P. asiatica from Korea.

• Journal of Nutrition and Health
• /
• v.55 no.2
• /
• pp.227-239
• /
• 2022

Purpose: This study investigated the effects of water-soluble mulberry leaf extract (ME) on hepatic lipid accumulation in high-fat diet-fed rats via the regulation of hepatic microRNA (miR)-221/222 and inflammation. Methods: Male Sprague-Dawley rats (4 weeks old) were randomly divided into 3 groups (n = 7 each) and fed with 10 kcal% low-fat diet (LF), 45 kcal% high-fat diet (HF), or HF + 0.8% ME for 14 weeks. Lipid profiles and cytokine levels of the liver and serum were measured using commercial enzymatic colorimetric and enzyme-linked immunosorbent assay, respectively. The messenger RNA (mRNA) and miR levels in liver tissue were assayed by real-time quantitative reverse-transcription polymerase chain reaction. Results: Supplementation of ME reduces body weight and improves the liver and serum lipid profiles as compared to the HF group. The mRNA levels of hepatic peroxisome proliferator-activated receptor-gamma, sterol regulatory element binding protein-1c, fatty acid synthase, and fatty acid translocase, which are genes involved in lipid metabolism, were significantly downregulated in the ME group compared to the HF group. In contrast, the mRNA level of hepatic carnitine palmitoyl transferase-1 (involved in fatty acid oxidation) was upregulated by ME supplementation. Furthermore, administration of ME significantly downregulated the mRNA levels of inflammatory mediators such as hepatic tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), monocyte chemoattractant protein-1, and inducible nitric oxide synthase. The serum levels of TNF-α, IL-6, and nitric oxide were also significantly reduced in ME group compared to the HF group. Expression of hepatic miR-221 and miR-222, which increase in the inflammatory state of the liver, were also significantly inhibited in the ME group compared to the HF group. Conclusion: These results indicate that ME has the potential to improve hepatic lipid accumulation in high-fat diet-fed rats via modulation of inflammatory mediators and hepatic miR-221/222 expressions.

• Journal of Life Science
• /
• v.32 no.8
• /
• pp.622-632
• /
• 2022

The purpose of this study was to investigate activities as functional cosmetic materials, focusing on Rhododendron brachycarpum (RB) and Rhododendron fortunei (RF). The tyrosinase inhibitory effect, related to skin-whitening, was 32.6% and 39.3% respectively at the concentration of 1,000 ㎍/ml. The elastase inhibitory effect, related to skin anti-wrinkling activity, was 30% and 36.2% at 1,000 ㎍/ml concentration. Collagenase inhibitory activity showed 77.7%, and 80.2% respectively at 1,000 ㎍/ml concentration, which demonstrated excellent inhibitory activity. For a cell viability test, that measured on fibroblast cells by RB and RF extracts. Furthermore, the cell viability test showed 100.9% and 98.9% with cell viability at 100 ㎍/ml concentration in CCD-986Sk. The protein expression inhibitory effect of RB and RF extracts was measured by western blot at 25, 50, and 100 ㎍/ml concentrations, and the β-actin was used as a positive control. As a result, western blot of RB and RF extracts was measured by the expression inhibition rate of the MMP-1, MMP-2, MMP-3 protein, and was decreased by 67.2%, 65.5%, 13.6%, and 89.1%, 85.0%, and 62.7% at 100 ㎍/ml concentration. The mRNA expression inhibitory effect of RB and RF extracts was measured by RT-PCR at 25, 50, and 100 ㎍/ml concentrations, and the GAPDH was used as a positive control. According to the results of RT-PCR of RB and RF extracts, the expression inhibition rate of the MMP-1, MMP-2, and MMP-3 mRNA was decreased by 70.1%, 9.1%, 37.9%, and 38.2%, 8.3%, 57.3% at 100 ㎍/ml concentrations. So, RB and RF extracts showed the anti-wrinkle effectiveness as a functional cosmetic material.