• Title/Summary/Keyword: reverse-transcription-PCR

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Sequence Analysis and Molecular Characterization of Wnt4 Gene in Metacestodes of Taenia solium

  • Hou, Junling;Luo, Xuenong;Wang, Shuai;Yin, Cai;Zhang, Shaohua;Zhu, Xueliang;Dou, Yongxi;Cai, Xuepeng
    • Parasites, Hosts and Diseases
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    • v.52 no.2
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    • pp.163-168
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    • 2014
  • Wnt proteins are a family of secreted glycoproteins that are evolutionarily conserved and considered to be involved in extensive developmental processes in metazoan organisms. The characterization of wnt genes may improve understanding the parasite's development. In the present study, a wnt4 gene encoding 491amino acids was amplified from cDNA of metacestodes of Taenia solium using reverse transcription PCR (RT-PCR). Bioinformatics tools were used for sequence analysis. The conserved domain of the wnt gene family was predicted. The expression profile of Wnt4 was investigated using real-time PCR. Wnt4 expression was found to be dramatically increased in scolex evaginated cysticerci when compared to invaginated cysticerci. In situ hybridization showed that wnt4 gene was distributed in the posterior end of the worm along the primary body axis in evaginated cysticerci. These findings indicated that wnt4 may take part in the process of cysticerci evagination and play a role in scolex/bladder development of cysticerci of T. solium.

Epidemiological Characterization of Influenza Viruses detected from Acute Respiratory Patients in Seoul during 2010-2012 (서울지역 급성호흡기 환자들로부터 분리한 인플루엔자 바이러스의 유행양상 (2010-2012))

  • Ham, Heejin;Jang, Jungim;Choi, Sungsun;Oh, Seah;Jo, Sukju;Choi, Sungmin;Pak, Sonil
    • Journal of Environmental Health Sciences
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    • v.39 no.3
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    • pp.230-238
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    • 2013
  • Objectives: For our survey of the incidence of influenza viruses among respiratory viral infections in Seoul, we evaluated their prevalence among infectious acute respiratory viral patients in Seoul from 2010 to 2012 through regular surveillance. Methods: For influenza virus detection, we conducted real-time PCR analyses on 2,544 throat specimens collected from patients with respiratory viral infections in Seoul between 2010 and 2012. They were collected and then tested for the presence of influenza viruses through reverse transcription (RT) - polymerase chain reaction (PCR). Results: 19.1% (486/2,544) of the throat specimens were determined to be positive for influenza viruses. The incidences of influenza viral infection in the case of respiratory viral infections through regular surveillance in Seoul were 23.0% (212/923) in 2010, 6.4% (47/738) in 2011, and 25.7% (227/883) in 2012, and 10.8% (275/2,544) of type A, and 8.3% (211/2,544) type B influenza viruses. In addition, the greatest prevalence was in the 20-49 age group (51.6% ), which shows that influenza viruses constituted a major causative agent of acute respiratory viral infections. Conclusions: The distributions of influenza viruses and the epidemiologic patterns of the viral pathogen in acute respiratory viral infectious patients may provide potentially effective data for epidemiological studies in Seoul, Korea.

Occurrence of Apple scar viroid-Korean strain (ASSVd-K) in Apples Cultivated in Korea

  • Lee, Ju-Hee;Park, Jean-Kyung;Lee, Dong-Hyuk;Uhm, Jae-Youl;Ghim, Sa-Youl;Lee, Jai-Youl
    • The Plant Pathology Journal
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    • v.17 no.5
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    • pp.300-304
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    • 2001
  • Apple is the most economically important fruit in Korea. The suspected viroid disease of dapple apple was found in apple fruits cultivated in Kyungpook province. Symptoms begin in mid-July as small circular spots, which stand out against the background color on the young fruit. Dappling of the fruit becomes more intense and easier to detect as the fruit approaches maturity; the affected spots remain yellowish as the fruit matures. no leaf or bark syndromes have been associated with this disease. The infected fruits are downgraded considerably during quality grading. The low molecular weight RNA containing viroid RNA molecules were extracted from the peels of the apples with dapple symptoms. The RNA molecules were extracted from the apples using Qiagen column chromatography. The purified RNAs were used for the synthesis of cDNA with RT-PCR. The PCR products were then ligated into a pGEM-T Easy vector, cloned and sequenced. The sequence of the viroid RNA molecule shows 331 nucleotides with one base difference ("G" insertion between the position of 133 and 134) compared with that of the Apple scar skin viroid (ASSVd) reported by Hashimoto and Koganezawa in Japan. This is the first report on the occurrence of the ASSVd in apple trees cultivated in Korea, as well as the identification of a new Korean strain of the ASSVd.the ASSVd.

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Effect of Mung Bean Lectin (MBL) on Cytokine Gene Expression from Human Peripheral Blood Mononuclear Cells (사람 말초혈액 단핵세포에서 녹두 렉틴의 사이토카인 생성효과)

  • Jeune, Kyung-Hee;An, Mong-Gi;Jung, Su-Min;Choi, Kyung-Min;Lee, Seung-Ho;Chung, See-Ryun
    • Korean Journal of Pharmacognosy
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    • v.30 no.4
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    • pp.355-362
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    • 1999
  • New lectins have been isolated and purified from mung bean (Phaseolus radiatus) through physiological saline extraction, ammonium sulfate salt fractionation and column chromatographies. Ion exchanger were eluted by linear salt gradient and then further purified through gel filtration. Thus obtained lectin named as MBL. The gene expressions of 5 cytokines (IL-1, IL-2, IL-6, $TNF-{\aphpa}$ and $IFN-{\gamma}$) from human peripheral blood mononuclear cells (PBMC) stimulated with MBL were investigated by using reverse transcription polymerase chain reaction (RT-PCR). PBMC ($1{\times}106$ cells/ml) isolated from healthy volunteers were stimulated with lectins (4 mg/ml) for various time intervals (1 to 96 hrs). After each of the various stimulated times, total RNA was isolated and assessed for different cytokines mRNA by RT-PCR. The mRNA encoding IL-1, IL-2 were detected continuously from 1 to 20 hrs, and IL-6 was detected up to 24 hrs. But the mRNA encoding $IFN-{\gamma}$ and $TNF-{\alpha}$ were detected to 8 hours only and showed short time response compared with other cytokines. The significant expression of all cytokines mRNA were observed at 4 hrs. These results suggested that MBL, as inducer of cytokines could elicit detectable cytokine mRNA from PBMC.

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First Report of Freesia sneak virus in Freesia spp. in Korea

  • Yoon, Ju-Yeon;Choi, Youn-Jung;Choi, Gug-Seoun;Choi, Seung-Kook
    • Research in Plant Disease
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    • v.19 no.4
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    • pp.313-318
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    • 2013
  • In March, 2013, twenty symptomatic freesia plants (10 plants of cultivar Shiny Lemon and 10 plants of cultivar Shiny Gold), with striking virus-like symptoms were collected in Cheongju, Korea. The plants showed chlorotic, coalescing, interveinal, whitish, necrotic, mosaic, mottling or dark brown-to-purple necrotic spots on leaves. Freesia crude sap was directly analyzed by transmission electron microscopy, which potyvirus particles as well as long virus-like particles were detected. Total RNA extracts were analyzed for the infection of Freesia sneak virus (FreSV) by reverse transcription (RT)-PCR with primers specific to FreSV coat protein (CP) gene based on the sequences of FreSV isolates (GenBank No. GU071089, FJ807730 and DQ885455), showing 9 of 20 plants were infected. All 1305bp RT-PCR products were cloned and sequenced. Comparisons of nucleotide and deduced amino acid sequences using BLAST and bioinformatics tools resulted in 99 to 100% sequence identity with FreSV isolates FOV, Virginia, and Italy, confirming FreSV in 9 symptomatic freesia plants. Of 9 determined cDNAs of FreSV isolates, sequences of 5 cDNA clones were identical (GenBank No. AB811437) and sequences of 4 cDNA clones were identical (GenBank No. AB811792). To our knowledge, this is the first report of FreSV from Freesia spp. in Korea.

Monitoring of Nervous Necrosis Virus (NNV) in the Broodstock of Seven Band Grouper Hyporthodus septemfasciatus (신경괴사증바이러스(Nervous Necrosis Virus, NNV) 모니터링을 통한 무감염 능성어(Hyporthodus septemfasciatus)친어의 선발)

  • Kim, Si-Woo;Kim, Wi-Sik;Seo, Han-Gill;Kim, Kyong Min;Oh, Myung-Joo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.50 no.5
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    • pp.527-533
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    • 2017
  • We investigated the infection of nervous necrosis virus (NNV) in seven band grouper Hyporthodus septemfasciatus broodstocks, which have been reared in aquaculture farms in South Korea during 2012-2014. To investigate the prevalence of NNV within the broodstock, egg, sperm, and blood were sampled in the spawning season. The egg and sperm samples were subjected to a nested reverse transcription (RT) polymerase chain reaction (PCR) assay to detect NNV and were inoculated on SSN-1 cells to culture the virus. Blood samples were used to detect antibodies against NNV using enzyme linked immunosorbent assay (ELSIA). Positive values from ELISA were found in 39 of 162 samples (24%) in 2012, and 13 of 28 samples (46%) in 2014. Additionally, 4 of 34 broodstocks (11%) investigated in 2013-2014 were determined to be carriers from the nested RT-PCR and in vitro cultivation. The broodstocks in which antibodies against NNV were detected by ELISA, or in which NNV was detected by the nested RT-PCR assay, posed a risk of vertical transmission of NNV. Therefore, it is necessary to select virus-free broodstocks in seed production to reduce the possibility of the vertical transmission of NNV.

Plasma Post-operative miR-21 Expression in the Prognosis of Gastric Cancers

  • Ma, Guo-Jian;Gu, Rong-Min;Zhu, Ming;Wen, Xu;Li, Jin-Tian;Zhang, Yuan-Ying;Zhang, Xiao-Mei;Chen, Sen-Qing
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.12
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    • pp.7551-7554
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    • 2013
  • Tumor-associated microRNAs have been detected in serum or plasma, but whether plasma microRNA-21 (miR-21) could be a potential circulating biomarker for gastric cancer (GC) prognosis in Chinese is still uncertain. Real-time quantitative reverse transcription PCR (qRT-PCR) was employed in this study to compare the relative expression of miR-21 between pre-operative and post-operative paired plasmas from 42 patients with primary GCs. The results showed that the expression levels of miR-21 in the post-operative plasmas were significantly reduced by an average of 18.2 times in all patients when compared to the pre-operative plasmas, and by 22.1 times in the subgroup of patients without family history, while only 1.76 times in the subgroup of patients with a family history. With respect of clinicopathological characteristics, the plasma miR-21 expression was highly associated with differentiation degree and lymph node metastasis rate. The results suggested plasma miR-21 could be a novel potential biomarker for GC prognosis and evaluation of surgery outcomes, especially in patients without a family history.

Effective Exon-Intron Structure Verification of a 1-Pyrroline-5-Carboxylate-Synthetase Gene from Halophytic Leymus chinensis (Trin.) Based on PCR, DNA Sequencing, and Alignment

  • Sun, Yan-Lin;Hong, Soon-Kwan
    • Korean Journal of Plant Resources
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    • v.23 no.6
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    • pp.526-534
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    • 2010
  • Genomes of clusters of related eukaryotes are now being sequenced at an increasing rate. In this paper, we developed an accurate, low-cost method for annotation of gene prediction and exon-intron structure. The gene prediction was adapted for delta 1-pyrroline-5-carboxylate-synthetase (p5cs) gene from China wild-type of the halophytic Leymus chinensis (Trin.), naturally adapted to highly-alkali soils. Due to complex adaptive mechanisms in halophytes, more attentions are being paid on the regulatory elements of stress adaptation in halophytes. P5CS encodes delta 1-pyrroline-5-carboxylate-synthetase, a key regulatory enzyme involved in the biosynthesis of proline, that has direct correlation with proline accumulation in vivo and positive relationship with stress tolerance. Using analysis of reverse transcription-polymerase chain reaction (RT-PCR) and PCR, and direct sequencing, 1076 base pairs (bp) of cDNA in length and 2396 bp of genomic DNA in length were obtained from direct sequencing results. Through gene prediction and exon-intron structure verification, the full-length of cDNA sequence was divided into eight parts, with seven parts of intron insertion. The average lengths of determinated coding regions and non-coding regions were 154.17 bp and 188.57 bp, respectively. Nearly all splice sites displayed GT as the donor sites at the 5' end of intron region, and 71.43% displayed AG as the acceptor sites at the 3' end of intron region. We conclude that this method is a cost-effective way for obtaining an experimentally verified genome annotation.

Upregulation and Clinicopathological Significance of Long Non-coding NEAT1 RNA in NSCLC Tissues

  • Pan, Lin-Jiang;Zhong, Teng-Fei;Tang, Rui-Xue;Li, Ping;Dang, Yi-Wu;Huang, Su-Ning;Chen, Gang
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.7
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    • pp.2851-2855
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    • 2015
  • Background: Recent reports have shown that nuclear enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA), contributes to the precise control of gene expression and is related to several human malignancies. However, limited data are available on the expression and function of NEAT1 in lung cancer. The major objective of the current study was to profile the expression and clinicopathological significance of NEAT1 in non-small cell lung cancers (NSCLCs). Materials and Methods: NEAT1 expression in 125 NSCLC cases and paired adjacent non-cancer tissues was assessed by real-time quantitative reverse transcription-PCR (qRT-PCR). Relationships between NEAT1 and clinicopathological factors were also investigated. Results: The relative level of NEAT1 was $6.98{\pm}3.74$ in NSCLC tissues, significantly elevated as compared to that of the adjacent non-cancer lung tissues ($4.83{\pm}2.98$, p<0.001). The area under curve (AUC) of high expression of NEAT1 to diagnose NSCLC was 0.684 (95% CI: 0.619~0.750, p<0.001). NEAT1 expression was positively correlated with patient age (r=-2.007, p=0.047), lymphatic metastasis (r=-2.731, p=0.007), vascular invasion (r=-3.617, p=0.001) and clinical TNM stage (r=-4.134, p<0.001). Conclusions: This study indicates that NEAT1 might be associated with oncogenesis and progression in NSCLC, and suggests application in molecular targeted therapy.

Characterization of the MicroRNA Expression Profile of Cervical Squamous Cell Carcinoma Metastases

  • Ding, Hui;Wu, Yi-Lin;Wang, Ying-Xia;Zhu, Fu-Fan
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.4
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    • pp.1675-1679
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    • 2014
  • Objectives: MicroRNAs (miRNAs) are important regulators of many physiological and pathological processes, including tumorigenesis and metastasis. In this study, we sought to determine the underlying molecular mechanisms of metastatic cervical carcinoma by performing miRNA profiling. Methods: Tissue samples were collected from ten cervical squamous cancer patients who underwent hysterectomy and pelvic lymph node (PLN) dissection in our hospital, including four PLN-positive (metastatic) cases and six PLN-negative (non-metastatic) cases. A miRNA microarray platform with 1223 probes was used to determine the miRNA expression profiles of these two tissue types and case groups. MiRNAs having at least 4-fold differential expression between PLN-positive and PLN-negative cervical cancer tissues were bioinformatically analyzed for target gene prediction. MiRNAs with tumor-associated target genes were validated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results: Thirty-nine miRNAs were differentially expressed (>4-fold) between the PLN-positive and PLN-negative groups, of which, 22 were up-regulated and 17 were down-regulated. Sixty-nine percent of the miRNAs (27/39) had tumor-associated target genes, and the expression levels of six of those (miR-126, miR-96, miR-144, miR-657, miR-490-5p, and miR-323-3p) were confirmed by quantitative (q)RT-PCR. Conclusions: Six MiRNAs with predicted tumor-associated target genes encoding proteins that are known to be involved in cell adhesion, cytoskeletal remodeling, cell proliferation, cell migration, and apoptosis were identified. These findings suggest that a panel of miRNAs may regulate multiple and various steps of the metastasis cascade by targeting metastasis-associated genes. Since these six miRNAs are predicted to target tumor-associated genes, it is likely that they contribute to the metastatic potential of cervical cancer and may aid in prognosis or molecular therapy.