• Title/Summary/Keyword: RT-PCR detection

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Spontaneous Release of Bacteriophage Particles by Lactobacillus rhamnosus Pen

  • Jarocki, Piotr;Podlesny, Marcin;Pawelec, Jaroslaw;Malinowska, Agata;Kowalczyk, Sylwia;Targonski, Zdzislaw
    • Journal of Microbiology and Biotechnology
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    • v.23 no.3
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    • pp.357-363
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    • 2013
  • The identification of bacteriophage proteins on the surface of Lactobacillus rhamnosus Pen was performed by LC-MS/MS analysis. Among the identified proteins, we found a phage-derived major tail protein, two major head proteins, a portal protein, and a host specificity protein. Electron microscopy of a cell surface extract revealed the presence of phage particles in the analyzed samples. The partial sequence of genes encoding the major tail protein for all tested L. rhamnosus strains was determined with specific primers designed in this study. Next, RT-PCR analysis allowed detection of the expression of the major tail protein gene in L. rhamnosus strain Pen at all stages of bacterial growth. The transcription of genes encoding the major tail protein was also proved for other L. rhamnosus strains used in this study. The present work demonstrates the spontanous release of prophage-encoded particles by a commercial probiotic L. rhamnosus strain, which did not significantly affect the bacterial growth of the analyzed strain.

Effects of Anemarrhenae Rhizoma on LPS-stimulated Expression of COX-2 and iNOS in mouse BV2 microglial cells

  • Seo, Bong-Won;Kim, Ee-Hwa;Park, Se-Keun;Jang, Mi-Hyeon;Choi, Sun-Mi;Lee, Eun-Yong
    • Journal of Acupuncture Research
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    • v.22 no.2
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    • pp.155-162
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    • 2005
  • Anemarrhenae Rhizoma (AR) has been widely used for the treatment of various diseases in Oriental medicine. To investigate whether AR possesses anti-inflammatory effects against lipopolysaccharide (LPS)-induced inflammation in the BV2 microglial cells, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. reverse transcription -polymerase chain reaction (RT-PCR), and prostaglandin E2 (PGE2) assay, and nitric oxide (NO) detection assay were performed. From the present results, AR was shown to suppress PGE2 synthesis and NO production by inhibiting the LPS-stimulated enhancement of cyclooxygenase-2 and inducible nitric oxide synthase expression in BV2 microglial cells. These results suggest that AR may offer a valuable means of therapy in the treatment of inflammatory diseases by attenuating LPS-induced PGE2 synthesis and NO production.

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The Use of a Tobacco mosaic virus-Based Expression Vector System in Chrysanthemum

  • Park, Minju;Baek, Eseul;Yoon, Ju-Yeon;Palukaitis, Peter
    • The Plant Pathology Journal
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    • v.33 no.4
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    • pp.429-433
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    • 2017
  • Chrysanthemums (Chrysanthemum morifolium) are susceptible to tobacco mosaic virus (TMV). TMV-based expression vectors have been used in high-throughput experiments for production of foreign protein in plants and also expressing green fluorescent protein (GFP) to allow visualization of TMV movement. Here, we used TMV expressing the GFP to examine the infection of chrysanthemum by a TMV-based expression vector. Viral replication, movement and GFP expression by TMV-GFP were verified in upper leaves of chrysanthemums up to 73 days post inoculation (dpi) by RT-PCR. Neither wild-type TMV nor TMV-GFP induced symptoms. GFP fluorescence was seen in the larger veins of the inoculated leaf, in the stem above the inoculation site and in petioles of upper leaves, although there was no consistent detection of GFP fluorescence in the lamina of upper leaves under UV. Thus, a TMV-based expression vector can infect chrysanthemum and can be used for the in vivo study of gene functions.

Effects of Sandalwood Essential Oil on the iNOS Expression and Proinflammatory Cytokine Production (Sandalwood Essential Oil의 iNOS 발현과 염증성사이토카인의 생성에 미치는 영향)

  • Park, Jeoung-Suk;Jung, Sook-Heui
    • YAKHAK HOEJI
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    • v.57 no.1
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    • pp.70-75
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    • 2013
  • The present study was designed to determine the effect of the Sandalwood Essential Oil (Santalum album) on pro-inflammatory factors such as NO, iNOS expression and IL-$1{\beta}$, IL-6, TNF-${\alpha}$ in lipopolysaccharide (LPS) - stimulated RAW264.7 macrophages cells. The cell toxicity was determined by MTS assay. To evaluate of anti-inflammatory effect of Sandalwood Essential Oil, amount of NO was measured using the NO detection kit and the iNOS expression was measured by western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). And proinflammatory cytokines were measured by ELISA kit. As a result, Sandalwood Essential Oil reduced NO, iNOS expression and IL-$1{\beta}$, IL-6, TNF-${\alpha}$ production without cytotoxicity. Our results suggest that the Sandalwood Essential Oil may have an anti-inflammatory property through suppressing inflammatory mediator productions and appears to be useful as an anti-inflammatory oil.

Production of Inflammatory Cytokines and Nitric Oxide by Human Mast Cells Incubated with Toxoplasma gondii Lysate

  • Park, Eun-Ah;Han, Ik-Hwan;Kim, Jung-Hyun;Park, Soon-Jung;Ryu, Jae-Sook;Ahn, Myoung-Hee
    • Parasites, Hosts and Diseases
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    • v.57 no.2
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    • pp.201-206
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    • 2019
  • The roles of mast cells in allergic diseases and helminth infections are well known. However, the roles of mast cells in T. gondii infection is poorly understood. This study was focused on the production of pro-inflammatory cytokines ($TNF-{\alpha}$, IL-4), chemokines (CXCL8, MCP-1) and nitric oxide (NO) by mast cells in response to soluble lysate of T. gondii tachyzoites. Production of CXCL8 (IL-8), MCP-1, $TNF-{\alpha}$ and IL-4 were measured by RT-PCR and ELISA. Western blot were used for detection of CXCR-1 and CXCR2. Our results showed that T. gondii lysates triggered mast cells to release CXCL8, MCP-1, $TNF-{\alpha}$, IL-4 and to produce NO. This suggests that mast cells play an important role in inflammatory responses to T. gondii.

Detection and phylogenetic analysis of norovirus from individual septic tanks in the drainage basin of the coastal area located in the Jaran Bay of Korea

  • Ham, In Tae;Kim, Byeo Ri;Park, Yu Jeong;Jung, Yeun Joong;Park, Kunbawui;Kwon, Ji Young;Mok, Jong Soo;Yu, Hongsik
    • Fisheries and Aquatic Sciences
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    • v.24 no.1
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    • pp.10-18
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    • 2021
  • Norovirus (NoV) prevalence was investigated in 100 sewage samples collected from 35 individual septic tanks around the drainage basin of Jaran Bay, Korea in January, May, and July of 2017. Genotypes and diversity of NoV strains detected in sewage samples were also assessed using the conventional RT-PCR and phylogenetic analysis. NoV GI or GII were detected in 22 (22.0%) and 24 (24.0%) samples, respectively. Thirteen genotypes were identified with three dominant genotypes (GI.9, GII.5 and GII.17) and GII.17 showed relatively higher prevalence during the survey period. GII.17 strains were clustered into recombinant type variant or NoV GII.17 Kawasaki variant. NoV GII.17 strains were considered emergent epidemic variants with widespread circulation. NoV surveillance strategy should include both environmental (sewage) and clinical data to reveal minor NoV genotypes likely cause of asymptomatic or underreported infections in the local population.

Incidence and variability of Hosta virus X and seed-transmission in Hosta plants

  • Park, M.H.;Lee, J.S.;K.H. Ryu
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.144.2-145
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    • 2003
  • This study was performed to investigate the incidence of Hosta uirus X (HVX), a Potexvirus, from cultivated hosta ornamental plants in Korea and to ascertain seed transmission of the virus from infected parent plant to progeny ones for breeding program of hosta plants. Infection rate of HVX in cultivated hostas was 25.6 % (11 out of 43 collected samples contained HVX) based on Western blot and RT-PCR detection methods. Most of HVX-infected hostas showed visible systemic leaf symptoms (mosaic, mottle, curling, stunting or combinations). Variability of HVX was confirmed by sequences of coat protein gene of individual isolates from different hostas. HVX was seed-transmitted on Hosta 'Blue Cadet'. The virus was detected from seeds, and sprouts and seedlings from the virus-contaminated seed sources. Over 7.5 % of seeds were HVX-contaminated surveyed in this study, Our data suggest that HVX can be transmitted by seed source, and indexing of the virus should be done for breeding program of Hosta.

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Surveillance of African swine fever infection in wildlife and environmental samples in Gangwon-do

  • Ahn, Sangjin;Kim, Jong-Taek
    • Korean Journal of Veterinary Service
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    • v.45 no.1
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    • pp.13-18
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    • 2022
  • African swine fever (ASF) is fatal to domestic pigs and wild boars (Sus scrofa) and affects the domestic pig industry. ASF is transmitted directly through the secretions of infected domestic pigs or wild boars, an essential source of infection in disease transmission. ASFV is also very stable in the environment. Thus, the virus is detected in the surrounding environment where ASF-infected carcasses are found. In this study, ASF infection monitoring was conducted on the swab and whole blood samples from wild animals, various hematopoietic arthropod samples that could access infected wild boar carcasses or habitats to cause maintenance and spread of disease, and soil samples of wild boar habitats. ASF viral DNA detection was confirmed negative in 317 wildlife and environmental samples through a real-time polymerase chain reaction. However, ASF occurs in the wild boars and spreads throughout the Korean peninsula. Therefore, it is necessary to trace the route of ASF virus infection by a continuous vector. Additional monitoring of various samples with potential ASF infection is needed to help the epidemiologic investigation and disease prevention.

Characterization of a Cell Line HFH-T2, Producing Viral Particles, from Primary Human Fetal Hepatocytes Infected with Hepatitis B Virus

  • Shim, Jae-Kyoung;Kim, Dong-Wook;Chung, Tae-Ho;Kim, June-Ki;Suh, Jeong-Ill;Park, Chun;Lee, Young-Choon;Chung, Tae-Wha;Song, Eun-Young;Kim, Cheorl-Ho
    • Journal of Microbiology and Biotechnology
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    • v.11 no.2
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    • pp.186-192
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    • 2001
  • A primary culture of human fetal hepatocytes was obtained through a therapeutic abortion process at 26 weeks of gestation period. More than $10^8$ cells were seeded on a plastic plate. These hepatocytes were infected with hepatitis B virus (HBV). The HBV was purified from serum of one chronic HBV carrier. Transformed hepatocytes were subcultured in a 10% FBS-supplemented medium. The morphology of the transformed cell was epithelial-like. The cells from the first pass showed signs of early proliferation and had a latent period of more than 3 months after 6-7 passages. After the rest period, the transformed cell proliferated actively and they were subcultured every three days. Transformed hepatocytes were characterized by detection of the HBV transcript by RT-PCR. The secretion of virions from transformed cells was investigated by PCR with the cell medium. Two types of virions secreted into the culture medium were examined by using the transmission electron microscope. Another approach to study the secretion of virions in to culture medium was carried out with HBV antibody. HBsAg was detected in the culture medium of transformed cells using ELISA and Western blot analyses. These data suggested that the human fetal hepatocyte cell line has been established by infection of HBV, in which this cell line secreted viral particles into the culture medium.

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Aberrant Methylation of RASSF2A in Tumors and Plasma of Patients with Epithelial Ovarian Cancer

  • Wu, Yu;Zhang, Xian;Lin, Li;Ma, Xiao-Ping;Ma, Ying-Chun;Liu, Pei-Shu
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.3
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    • pp.1171-1176
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    • 2014
  • Objective: The tumor suppressor gene, Ras-association domain family (RASSF)2A, is inactivated by promoter hypermethylation in many cancers. The current study was performed to evaluate the methylation status of RASSF2A in epithelial ovarian cancer (EOC) tissues and plasma, and correlations with gene expression and clinicopathologic characteristics. Method: We detected methylation of the RASSF2A gene in tissues and corresponding plasma samples from 47 EOC patients and 14 patients with benign ovarian tumors and 10 with normal ovarian tissues. The methylation status was determined by methylation-specific PCR while gene expression of mRNA was examined by RT-PCR. The EOC cell line, SKOV3, was treated with 5-aza-2'-deoxycytidine (5-azadC). Results: RASSF2A mRNA expression was significantly low in EOC tissues. The frequency of aberrant methylation of RASSF2A was 51.1% in EOC tissues and 36.2% in corresponding plasma samples, whereas such hypermethylation was not detected in the benign ovarial tumors and normal ovarian samples. The expression of RASSF2A mRNA was significantly down-regulated or lost in the methylated group compared to the unmethylated group (p<0.05). After treatment with 5-aza-dC, RASSF2A mRNA expression was significantly restored in the Skov3 cell line. Conclusion: Epigenetic inactivation of RASSF2A through aberrant promoter methylation may play an important role in the pathogenesis of EOC. Methylation of the RASSF2A gene in plasma may be a valuable molecular marker for the early detection of EOC.