• Title/Summary/Keyword: PCR (Polymerase Chain Reaction)

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Identification of Papaya Ringspot Potyvirus type W infecting squash in Korea

  • T. S. Jin;Lee, S. H.;Park, J. W.;Park, H.S.;Kim, M.;D. B. Shin;J. U. Cheon;B. J. Cha
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.141.2-142
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    • 2003
  • A flexuous rod-shaped virus was isolated from Cucurbita pepo leaves showing green mosaic and puckering symptoms at Anseong, Korea. Based on the biological tests, electron microscopy, and reverse transcription-polymerase chain reaction (RT-PCR), the isolate was identified as Papaya ringspot virus type Watermelon (PRSV-W). In the biological test, host range of PRSV-W was limited in the families Cucurbitaceae and Chenopodiaceae. Most susceptible cucurbit species, such as Cucurmis lanatus, Cucurmis sativus, Cucurbita pepo, and Citrullus lanatus, responded to mechanical inoculation by PRSV-W that induce green mosaic, malformation, puckering, and narrow laminae. The local lesion symptoms were produced on the inoculated leaves of Chenopodium maranticolor and C. quinoa PRSV specific primers which amplifies the part of the coat protein (CP) genes, generated a 648 bp product from 6 isolates of PRSV-W, but no amplification had been detected in other viruses including CMV, CGMMV, KGMMV, ZYMV and WMV. In electron microscopy, PRSV particles were flexuous, approximately 780 nm in length and 12 nm in width. PRSV-W is one of the worldwide viruses which has the great economic importance in cucumber, melon, squash, watermelon, and other cultivated cucurbits with ZYMV and WMV. This is the first report of PRSV-W on cucurbits in Korea.

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Effects of topical application of Phospholipid derivatives on the secretion of sebum on the skin of the fuzzy rats

  • Y. A. Hwang;Park, W. K.;Park, C. Y.;Kim, J. W.;Park, C. S.
    • Proceedings of the SCSK Conference
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    • 2003.09a
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    • pp.578-589
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    • 2003
  • The fuzzy rat that expresses hypersecretion of sebum and hyperplastic sebaceous glands is a genetic mutant for the study of many pharmacological aspects especially human acne. Through this model, we examined the effects of several phospholipids on the secretion of sebum after topical application. The phospholipid derivatives were phosphatidylcholine (PC), hydrogenated phosphatidylcholine (HPC), phosphati dylserine (PS) and hydrogenated phosphatidylserine(HPS). All agents were dissolved into the vehicle (1, 3-Butanediol, ethanol and water) at 0.5% weight volume and applied on the dorsal area of the fuzzy rat. To observe histological changes, the skin biopsies were stained with Oil Red O and the size and morphology of sebaceous gland was observed under microscope. Topical treatment with PC and/or HPC showed a marked decrease in sebum excretion. Especially hydrogenated PC (HPC) appeared to have more predominant sebosuppressive function than any other treatment. The other agents such as PS and HPS showed a marginal effect on sebum secretion. With the sebosuppressive activity, HPC and PC seem to have a good potential application on acne treatment. In order to obtain more insights into possible mechanisms behind the above observations, effects of each phospholipid on the expression of peroxisome proliferator-activated receptor (PPAR) genes were investigated. Recently, it has been demonstrated that expression and activation of PPAR subtypes appear to modulate the accumulation of cytoplasmic fat droplets that characterizes the sebocyte differentiation(1). It was also previously suggested that PPAR${\gamma}$ antagonist would seem possible to interfere sebum production without side effects (2). In this study we examined the diverse effects of the tested phospholipids on the expression of several PPAR genes based on reverse transcription-polymerase chain reaction (RT-PCR) from the topically treated skin of fuzzy rats. The results and possible implications are discussed.

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Characterization of Vancomycin Resistant Enterococci and Drug Ligand Interaction between vanA of E. faecalis with the Bio-Compounds from Aegles marmelos

  • Jayavarsha V;Smiline Girija A.S;Shoba Gunasekaran;Vijayashree Priyadharsini J
    • Journal of Pharmacopuncture
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    • v.26 no.3
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    • pp.247-256
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    • 2023
  • Objectives: Enterococcus faecalis is a gram positive diplococci, highly versatile and a normal commensal of the gut microbiome. Resistance to vancomycin is a serious issue in various health-care setting exhibited by vancomycin resistant Enterococci (VRE) due to the alteration in the peptidoglycan synthesis pathway. This study is thus aimed to detect the VRE from the patients with root caries from the clinical isolates of E. faecalis and to evaluate the in-silico interactions between vanA and the Aegles marmelos bio-compounds. Methods: E. faecalis was phenotypically characterized from 20 root caries samples and the frequency of vanA and vanB genes was detected by polymerase chain reaction (PCR). Further crude methanolic extracts from the dried leaves of A. marmelos was assessed for its antimicrobial activity. This is followed by the selection of five A. marmelos bio-compounds for the computational approach towards the drug ligand interactions. Results: 12 strains (60%) of E. faecalis was identified from the root caries samples and vanA was detected from two strains (16%). Both the stains showed the presence of vanA and none of the strains possessed vanB. Crude extract of A. marmelos showed promising antibacterial activity against the VRE strains. In-silico analysis of the A. marmelos biocompounds revealed Imperatonin as the best compound with high docking energy (-8.11) and hydrogen bonds with < 140 TPSA (Topological polar surface area) and zero violations. Conclusion: The present study records the VRE strains among the root caries with imperatorin from A. marmelos as a promising drug candidate. However the study requires further experimentation and validation.

Copper Regulates Apelin Expression in L6 Skeletal Muscle Cells (골격근세포에서 구리에 의한 마이오카인 apelin의 발현)

  • Kisang Kwon;Jin Sol Park;Young Eun Choi;Eun-Ryeong Lee;Jae Eun Yoo;Hyewon Park;O-Yu Kwon
    • Journal of Life Science
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    • v.33 no.9
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    • pp.724-729
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    • 2023
  • In this experiment, we aimed to investigate the role of copper in regulating the biosynthesis of a myokine called apelin in mammalian skeletal muscle cells. Our approach involved culturing skeletal muscle cells and subjecting them to treatments with copper sulfate or a copper chelator known as bathocuproinedisulfonic acid (BCS). We employed standard techniques, such as reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, to assess the synthesis of apelin at different stages, including transcription, translation, and post-translational modifications. Our findings demonstrated that copper had an inhibitory effect on apelin biosynthesis at all three stages: transcription, translation, and post-translation. However, when we treated the cells with BCS, the biosynthesis of apelin was restored to its original state. This finding suggests that copper is required for the synthesis of apelin in mammalian skeletal muscle cells. This study represents the first documented evidence of the inorganic copper-dependent regulation of apelin biosynthesis, shedding light on potential strategies for the prevention and treatment of sarcopenia induced by copper imbalances.

Molecular Marker Development for the Rapid Differentiation of Black Rot Causing Xanthomonas campestris pv. campestris Race 7

  • Yeo-Hyeon Kim;Sopheap Mao;Nihar Sahu;Uzzal Somaddar;Hoy-Taek Kim;Masao Watanabe;Jong-In Park
    • The Plant Pathology Journal
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    • v.39 no.5
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    • pp.494-503
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    • 2023
  • Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen of Brassica crops that causes black rot disease throughout the world. At present, 11 physiological races of Xcc (races 1-11) have been reported. The conventional method of using differential cultivars for Xcc race detection is not accurate and it is laborious and time-consuming. Therefore, the development of specific molecular markers has been used as a substitute tool because it offers an accurate and reliable result, particularly a quick diagnosis of Xcc races. Previously, our laboratory has successfully developed race-specific molecular markers for Xcc races 1-6. In this study, specific molecular markers to identify Xcc race 7 have been developed. In the course of study, whole genome sequences of several Xcc races, X. campestris pv. incanae, X. campestris pv. raphani, and X. campestris pv. vesicatoria were aligned to identify variable regions like sequence-characterized amplified regions and insertions and deletions specific to race 7. Primer pairs were designed targeting these regions and validated against 22 samples. The polymerase chain reaction analysis revealed that three primer pairs specifically amplified the DNA fragment corresponding to race 7. The obtained finding clearly demonstrates the efficiency of the newly developed markers in accurately detecting Xcc race 7 among the other races. These results indicated that the newly developed marker can successfully and rapidly detect Xcc race 7 from other races. This study represents the first report on the successful development of specific molecular markers for Xcc race 7.

DNA Array Analysis of Changes in Gene Expression Profile in DHEA-induced PCO

  • Yu, Jeong-Min;Yoo, Seong-Jin;Kim, Do-Rim;Youm, Mi-Young, Kim, Jee-Yun;Kang, Sung-Goo
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.112-112
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    • 2002
  • Under normal conditions, women produce a single dominant follicle that participates in a single ovuation each menstrual cycle. But Polycystic ovary syndrome(PCOS) conditions, folliculogenesis does not proceed normally. This condition leads to the accumlation of large numbers of small graffian follicles in which the theca interstitial cells (TIC) produce abnormally large amounts of androgen. PCOS is probably the most common endocrine disorder, affecting women of reprodutive age with 5-10% prevalence estimate. Chronic anovulation, hyperandrogenism, hirsutism, obesity, infertility and polycystic ovaries are clinical hallmarks of women with PCOS. Its etiology remains unknown. To investigate the gene expression pattern of ovary in PCO-induced rat, we used cDNA expression analysis. Total RNA was extracted from the ovary of PCO-induced rat and reverse-transcribed in the presence of[$\alpha$$^{32}$P]-dATP Which were hybridized to Atlas$^{TM}$ Rat Toxicology 1.2 array (Clontech) representing approximately 1176 rat genes. We compared gene expression between ovary of pco-induced immature female rats and control. Differential gene expression profiles were revealed (LIFR-alpha, ADRA1A, Heat shock 90-kDa protein A, PDGFRA). Reverse transcription-polymerase chain reaction(RT-PCR) was used to validate the relative expression pattern obtained by the cDNA array. The precise relationship between the altered expression of genes and PCO is a matter of further investigation. This study was supported by Korea Science and Engineering Foundation(KOSEF)

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5,10-Methylenetetrahydrofolate reductase 677C>T polymorphism and microsatellite instability in sporadic colorectal cancer (산발성 현미부수체 불안정성 대장암의 임상적 의의 및 MTHFR 677C>T 유전자 다형성과의 관계)

  • Kwon, Su-kyung;Kim, Jong Woo;Kim, Nam Keun
    • Korean Journal of Clinical Oncology
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    • v.9 no.2
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    • pp.80-86
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    • 2013
  • Purpose: Hypermethylation of human mut L homologue 1 (hMLH1) promoter region is known to cause sporadic microsatellite instability (MSI) colorectal cancers. 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in folate metabolism, acting as a methyl donor for DNA methylation. In this study, we investigate whether the polymorphism of MTHFR 677C>T plays a role in the alteration of the promoter-specific hypermethylation, predisposing to MSI colorectal cancers. Methods: Total of 487 sporadic colorectal cancer patients in CHA Bundang Medical Center were collected. MSI was identified when two or more are positive among five microsatellite markers (BAT25, BAT26, D17S250, D5S346, D2S123). The others were classified as microsatellite stable (MSS). Polymorphism of MTHFR 677C>T was genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: MSI was observed in 65 of 487 patients (12.73%). MSI colorectal cancers showed similar clinicopathological features with previously reported; younger age onset, right-sided preponderance, mucinous and poorly differentiated histology, lower stage, fewer lymph node metastases than MSS tumors (each P<0.05). The frequency of MTHFR 677TT genotype was 17.7% in the MSI group higher than 14.6% in the MSS group (P=0.17). Although not statistically significant, compared to the MTHFR 677CC referent, MTHFR 677 CT+TT genotype was more likely to have MSI than MSS (odds ratio, 1.81; 95% confidence interval, 0.94 to 3.68; P=0.06). Conclusion: This study demonstrated higher frequency of MTHFR 677TT genotype in MSI colorectal cancers. Furthermore, individuals with MTHFR 677CT+TT variant type might potentially develop MSI rather than MSS colorectal cancers.

Culturing characteristics of Hanwoo myosatellite cells and C2C12 cells incubated at 37℃ and 39℃ for cultured meat

  • Sehyuk Oh;Sanghun Park;Yunhwan Park;Yun-a Kim;Gyutae Park;Xiangshun Cui;Kwansuk Kim;Seontea Joo;Sunjin Hur;Gapdon Kim;Jungseok Choi
    • Journal of Animal Science and Technology
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    • v.65 no.3
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    • pp.664-678
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    • 2023
  • To improve culture efficiency of Hanwoo myosatellite cells, these cells were cultured at different temperatures. Hanwoo myosatellite cells were compared with C2C12 cells to observe proliferation and differentiation at culture temperatures of 37℃ and 39℃ and determine the possibility of using them as cultured meat. Immunofluorescence staining using Pax7 and Hoechst, both cells cultured at 37℃ proliferated better than cultured at 39℃ (p < 0.05). When differentiated cells were stained with myosin and Hoechst, there was no significant difference in myotube thickness and Fusion index (p > 0.05). In Western blotting analysis, Hanwoo myosatellite cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). C2C12 cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). In reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis, Hanwoo myosatellite cells cultured at 39℃ had significantly (p < 0.05) higher expression levels of MyHC, MYF6, and MB than those cultured at 37℃. C2C12 cells cultured at 39℃ showed significantly (p < 0.05) higher expression levels of MYOG and MB than those cultured at 37℃. To increase culture efficiency of Hanwoo myosatellite cells, proliferating at 37℃ and differentiating at 39℃ are appropriate. Since results of temperature differences of Hanwoo myosatellite cells were similar to those of C2C12 cells, they could be used as a reference for producing cultured meat using Hanwoo satellite cells.

Anti-inflammation Effect of Cyrtomium fortunei J.Sm. Extracts in Lipopolysaccharides-induced Microglia BV2 Cell (LPS로 자극한 microglia BV2 cell에서 Cyrtomium fortunei J.Sm. 추출물의 항염증 효과)

  • Jiwon Choi;Shintae Kim;Sang Yoon Choi;Inwook Choi;Jinyoung Hur
    • Journal of the Korean Society of Food Culture
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    • v.38 no.3
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    • pp.176-183
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    • 2023
  • In this study, we investigated the effect of the extracts of Cyrtomium fortunei J.Sm. (CFJ) on lipopolysaccharide (LPS) induced inflammation in mouse BV-2 microglial cells. Nitric oxide (NO) production and cell viability were measured using the Griess reagent and the (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay. Inflammatory cytokines were detected by quantitative polymerase chain reaction (qPCR) in BV-2 microglial cells with and without CFJ extracts. Subsequently, mitogen-activated protein kinases (MAPKs) and antioxidant markers were assessed by western blot analysis. It was found that the CFJ extract significantly decreased the production of pro-inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-1β) and NO in BV-2 microglial cells that were stimulated with LPS. In addition, the expression levels of the phosphorylation of the MAPK family (p38, c-Jun N-terminal kinases [JNK], and extracellular-signal regulated kinase [ERK]) were reduced by CFJ. Also, treatment with CFJ significantly increased the activities of superoxide dismutase type 1(SOD1) and Catalase in BV-2 microglial cells. Our results indicate that CFJ has a potent suppressive effect on the pro-inflammatory responses of activated BV-2 microglia. Therefore, CFJ has the potential to be an effective treatment for neurodegenerative diseases, as it can inhibit the production of inflammatory mediators in activated BV-2 microglial cells.

Relationship between angiotensin-converting enzyme gene polymorphism and muscle damage parameters after eccentric exercise

  • Kim, Jooyoung;Kim, Chang-Sun;Lee, Joohyung
    • Korean Journal of Exercise Nutrition
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    • v.17 no.2
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    • pp.25-34
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    • 2013
  • This study was conducted to investigate the relationship between ACE gene polymorphism and muscle damage parameters after eccentric exercise. 80 collegiate males were instructed to take an eccentric exercise with the elbow flexor muscle through the modified preacher curl machine for 2 sets of 25 cycles (total 50 cycles). The maximal isometric strength, muscle soreness, creatine kinase (CK), and myoglobin (Mb) were measured before exercise, and 0, 24, 48, 72, and 96 hrs after exercise. The result showed that after the eccentric exercise, the maximal isometric strength significantly decreased by more than 50% (p < 0.001) and the muscle soreness, CK, and Mb significantly increased compared to those before the exercise (p < 0.001). The ACE gene polymorphism of the subjects was classified using real-time polymerase chain reaction (real-time PCR). The result showed that it consisted of 38 cases of type II (46.4%), 33 cases of type ID (43.4%), and 9 cases of type DD (10.2%). The Hardy-Weinberg equilibrium for ACE gene polymorphism was shown to have p = 0.653, which showed that each allele was evenly distributed. Although significant differences in the changes in the maximal isometric strength, muscle soreness, CK, and Mb were found according to time course (p < 0.001), no significant differences in the changes in the maximal isometric strength, muscle soreness, CK, and Mb were found according to ACE gene polymorphism. Furthermore, no significant difference in the changes in the muscle damage parameters was found according to interaction between ACE gene polymorphism and time course (p > 0.05). In conclusion, the level of the muscle damage parameters changed in the injured muscle after eccentric exercise, but these changes in the muscle damage parameters were not affected by ACE gene polymorphism. The result of this study indicates that ACE gene is not a candidate gene that explains muscle damage.