• Title/Summary/Keyword: CRT-PCR

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Quantitation of Hepatitis C Viral RNA Using Direct CRT-PCR

  • Park, Young-Suk;Lee, Kyung-Ok;Oh, Moon-Ju;Chai, Young-Gyu
    • BMB Reports
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    • v.30 no.3
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    • pp.234-236
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    • 1997
  • Chronic hepatitis C virus (HCV) infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. It has been reported that the amount of HCV RNA may be correlated with the progression of hepatitis and may be a prognostic marker for treatment of HCV patients. The direct detection of HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. In the present study, we developed the method for HCV quantitation using competitive reverse transcription (CRT)-PCR using the deleted HCV standard. The serially diluted standard was added in titrated amounts to the target HCV RNA. The mixture was then reverse transcribed and amplified in the same reaction tube. The methods were evaluated using over 110 HCV-PCR positive samples in Koreans. About 59% of the samples were judged to contain $10^{5}-10^{6}$ copies of HCV RNA in 1 ml of serum.

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Impact of Time Interval Between Chemoradiation and Surgery on Pathological Complete Response and Survival in Rectal Cancer

  • Akbar, Ali;Bhatti, Abu Bakar Hafeez;Niazi, Samiullah Khan;Syed, Amir Ali;Khattak, Shahid;Raza, Syed Hassan;Kazmi, Ather Saeed
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.1
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    • pp.89-93
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    • 2016
  • Background: Limited data are available regarding the impact of time duration between chemoradiation (CRT) and surgery on pathological complete response (PCR). A PCR translates into better overall and disease free survival. The objective of this study was to determine effect of time duration on outcome after preoperative CRT in rectal cancer. Materials and Methods: A retrospective review of patients undergoing operations for rectal adenocarcinoma between January 2005 and December 2010 was performed. Patients were divided in two groups: Group 1 underwent surgery in ${\leq}8weeks$ post neoadjuvant CRT and Group 2 after 8 weeks. Patient characteristics, surgical procedure, histopathological details and number of loco-regional and distant failures were compared. Expected 5 year overall survival and disease free survival was calculated using Kaplan Meier curves and significance was determined using the log rank test. Results: There were 66 patients in group 1 and 93 in group 2. No significant difference in PCR was observed between the two. However, estimated 5 year DFS was significantly higher in Group 1 (66.7%) as compared to Group 2 (53.8%) (P=0.04). Estimated overall 5 year overall survival was not significantly different at 68.2% versus 54.3% (P= 0.09). Conclusions: Delaying surgery more than 8 weeks after preoperative CRT does not impact for PCR in rectal cancer.

In vitro immunoregulatory role of recombinant Ancylostoma ceylanicum calreticulin

  • Tingting Zhuang;Asmaa M. I. Abuzeid;Xiaoyu Chen;Shilan Zhu;Guoqing Li
    • Parasites, Hosts and Diseases
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    • v.62 no.1
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    • pp.75-84
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    • 2024
  • Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestines of humans and animals (dogs and cats), leading to malnutrition and iron-deficiency anemia. Helminth parasites secrete calreticulin (CRT), which regulates or blocks the host's immune response. However, no data on A. ceylanicum calreticulin (Ace-CRT) are available. We investigated the biological function of recombinant Ace-CRT (rAce-CRT). rAce-CRT showed reliable antigenicity and stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells. Quantitative reverse-transcription PCR assays revealed that rAce-CRT primarily promoted the expression of T helper 2 cytokines, particularly IL-13, in canine peripheral blood lymphocytes. rAce-CRT inhibited complement-mediated sheep erythrocyte hemolysis in vitro. Our findings indicate that Ace-CRT plays an immunomodulatory role and may be a promising candidate molecule for a hookworm vaccine.

Suppression of tobamovirus movement toward upper leaves in the tomato plant over-expressing a maize calreticulin (옥수수 calreticulin 과발현 토마토에서 tobamovirus의 상엽 이동 억제)

  • Han, Jeung-Sul
    • Journal of Plant Biotechnology
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    • v.37 no.4
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    • pp.567-573
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    • 2010
  • To ascertain the effect of over-expressed maize calreticulin in tomato plant on tobamovirus movement in addition to validating potentiality of the gene (ZmCRT) as a means for the virus-resistance resource, four ZmCRT-expressing homozygous lines were generated from the T0 plants as using an Agrobacterium-mediated transformation, nucleic acid analyses, and a conventional breeding method. Of them, a line was subjected to the bioassay for tolerances to tobacco mosaic virus-U1 (TMV-U1) and tomato mosaic virus (ToMV) followed by RT-PCR and a chlorophyll fluorescence quenching analyses. Both transgenic plants transcribing ZmCRT and wild-type plants showed no symptom by 20 days after viruses inoculation, however the photosystem II quantum yield parameter measured from the upper leaves of ToMV-inoculated plants revealed that ZmCRT transgenic plants have higher photosynthetic ability than wild-type ones at that time, which indirectly implies that over-expressed ZmCRT product acts as a barrier to the cell-to-cell and/or systemic movement of ToMV. Moreover, ZmCRT transgenic plants showed remarkably longer shoot length than wild-type ones in 40 days after TMV-U1 or ToMV inoculation each, which might be resulted from higher photosynthetic ability during the phase not yet showing any external symptoms. Collectively, over-expressed ZmCRT protein in tomato plants is able to interrupt the systemic movement of infected TMV-U1 and ToMV even though not perfect.

A Gene-based dCAPS Marker for Selecting old-gold-crimson (ogc) Fruit Color Mutation in Tomato (토마토 과색 돌연변이 유전자(old-gold-crimson) 선발을 위한 dCAPS 분자표지 개발)

  • Park, Young-Hoon;Lee, Yong-Jae;Kang, Jum-Soon;Choi, Young-Whan;Son, Beung-Gu
    • Journal of Life Science
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    • v.19 no.1
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    • pp.152-155
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    • 2009
  • The old-gold-crimson ($og^c$) fruit color mutation produces deep red tomato fruit with high lycopene content. age is a null mutation allele of lycopene-${\beta}$-cyclase (Crt-b) gene (B locus) that converts lycopene to ${\beta}$-carotene in the cartenoid biosynthesis pathway in tomato. Breeding of high lycopene tomato cultivars can be accelerated by marker-assisted selection (MAS) for introgression of $og^c$ allele by using a gene-based DNA marker. In order to develop a marker, single nucleotide deletion of adenine(A) with. in a poly-A repeat that has been known to be responsible for frame-shift mutation of $og^c$ was confirmed by resequencing mutant allele and wild-type allele at B locus of several tomato lines. For allele discrimination and detection of $og^c$, derived CAPS (dCAPS) approach was used by designing a primer that artificially introduced restriction enzyme recognition site of Hin fI in PCR products from $og^c$ allele. This dCAPS marker is co-dominant gene-based PCR marker that can be efficiently used for MAS breeding program aiming the development of high lycopene tomato.

LC-MS/MS-based Proteomic Analysis of Locally Advanced Rectal Tumors to Identify Biomarkers for Predicting Tumor Response to Neoadjuvant Chemoradiotherapy

  • Kim, Kyung-Ok;Duong, Van-An;Han, Na-Young;Park, Jong-Moon;Kim, Jung Ho;Lee, Hookeun;Baek, Jeong-Heum
    • Mass Spectrometry Letters
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    • v.13 no.3
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    • pp.84-94
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    • 2022
  • Neoadjuvant chemoradiotherapy (nCRT) is a standard therapy used for locally advanced rectal cancer prior to surgery, which can more effectively reduce the locoregional recurrence rate and radiation toxicity compared to postoperative chemoradiotherapy. The response of patients to nCRT varies, and thus, robust biomarkers for predicting a pathological complete response are necessary. This study aimed to identify possible biomarkers involved in the complete response/non-response of rectal cancer patients to nCRT. Comparative proteomic analysis was performed on rectal tissue samples before and after nCRT. Proteins were extracted for label-free proteomic analysis. Western blot and real-time PCR were performed using rectal cancer cell line SNU-503 and radiation-resistant rectal cancer cell line SNU-503R80Gy. A total of 135 up- and 93 down-regulated proteins were identified in the complete response group. Six possible biomarkers were selected to evaluate the expression of proteins and mRNA in SNU-503 and SNU-503R80Gy cell lines. Lyso-phosphatidylcholine acyltransferase 2, annexin A13, aldo-ketose reductase family 1 member B1, and cathelicidin antimicrobial peptide appeared to be potential biomarkers for predicting a pathological complete response to nCRT. This study identified differentially expressed proteins and some potential biomarkers in the complete response group, which would be further validated in future studies.

Quantitative RT-PCR for Measuring C-fos Gene Expression in Rat Brain after ECS (전기경련충격시 경쟁적 역전사 중합효소연쇄반응(CRT-PCR)을 이용한 흰쥐 뇌 c-fos 유전자의 발현 양식 분석)

  • Yang, Byung-Hwan;Lee, Jei-Wook;Park, Eung-Chul;Yu, Jae-Hak;Cho, Goang-Won;Yang, Bo-Gee;Chai, Young-Gyu
    • Korean Journal of Biological Psychiatry
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    • v.3 no.2
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    • pp.181-190
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    • 1996
  • To clarify the mechanism of action of electroconvulsive shack(ECS) in respect to molecular biology, and to detect the quantitative amount of change of c-fos gene expression after ECS in the rat's brain, the authors obtained brain specimens from the striatum, cerebral cortex, hippocampus, and cerebellum. Each brain was removed within 30min. after ECS(130V, 0.5sec) and ECS-sham. Then we performed RT-PCR. The results are 1) ECS was found to affect the expression of immediate early genes. 2) the cerebral cortex and hippocampus was more influenced by ECS thon in the cerebellum and striatum. From these results, we can suggest that ECS is related to the mechanism of cognition, mood, memory which is correlated to cerebral cortex and hippocampus.

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Heterologous expression of the Arabidopsis DREB1A/CBF3 gene enhances drought and freezing tolerance in transgenic Lolium perenne plants

  • Li, Xue;Cheng, Xiaoxia;Liu, Jun;Zeng, Huiming;Han, Liebao;Tang, Wei
    • Plant Biotechnology Reports
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    • v.5 no.1
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    • pp.61-69
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    • 2011
  • The dehydration-responsive element binding proteins (DREB1)/C-repeat (CRT) binding factors (CBF) function as transcription factors and play an important role in agricultural biotechnology and molecular biology studies of drought and freezing stress tolerance. We generated transgenic Lolium perenne plants containing the PCR-cloned Arabidopsis DREB1A/CBF3 gene (AtDREB1A/CBF3) to study the function of this gene construct in drought and freezing tolerance in a species of turfgrass. Compared to the control, AtDREB1A/CBF3 transgenic L. perenne plants showed enhanced drought and freezing stress tolerance. The activities of the enzymes superoxide dismutase (SOD) and peroxidase (POD) were higher in transgenic plants than in the non-transgenic plant control. These results demonstrate that the expression of the AtDREB1A/CBF3 gene in transgenic L. perenne plants enhanced drought and freezing tolerance and that the increased stress tolerance was associated with the increased activities of antioxidant enzymes. These results are relevant to stress biology and biotechnology studies of turfgrass.

Cloning And Characterization of Pathogen-Inducible EREBP-Like Transcription Factor(CaNR19) From Hot Pepper (Capsicum annuum L.)

  • Yi, So-Young;Kim, Jee-Hyub;Yu, Seung-Hun;Park, Doil
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.77.2-78
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    • 2003
  • An EREBP/AP2-type transcription factor (CaPFl) was isolated by DDRT-PCR following inoculation of soybean pustule pathogen Xanthomonas axonopodis pv. glycines Bra which induces HR on pepper leaves. Genomic Southern blot analysis revealed that the CaPFl gene is present as a single copy within the hot pepper genome. The deduced amino acid sequence of CaPFl has two potential nuclear localization signals, a possible acidic activation domain, and an EREBP/AP2 motif that could bind to a conserved cis- element present in promoter region of many stress-induced genes. The mRNA level of CaPFl was induced by both biotic and abiotic stresses. We observed higher-level transcripts in resistance-induced pepper tissues than diseased tissues. Expression of CaPFl is also induced upon various abiotic stresses including ethephon, MeJA, cold stress, drought stress and salt stress treatments. To study the role of CPFI in plant, transgenic Arabidopsis and tobacco plants which express higher level of pepper CaPFl were generated. Global gene expression analysis of transgenic Arabidopsis by cDNA microarray indicated that expression of CaPFl in transgenic plants affect the expression of quite a few GCC box and DRE/CRT box-containing genes. Furthermore, the transgenic Arabidopsis and tobacco plant, expressing CaPFl showed tolerance against freezing temperature and enhanced resistance to Pseudomonas syrnigae pv. tabaci. Taken together, these results indicated that CaPFl is a novel EREBP/AP2 transcription factor in hot pepper plant and it may has a significant role(s) in regulation of biotic and abiotic stresses in plant.

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