• Title/Summary/Keyword: Real-Time polymerase chain reaction (PCR)

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Altered Expression of ${\beta}_3$ Integrin on Sclerotic Aortic Valves in a Hypercholesterolemic Rabbit Model (고콜레스테롤혈증을 유발한 토끼의 대동맥 판막에서 ${\beta}_3$ Integrin 발현의 변화)

  • Park, Chan-Beom;Kim, Young-Du;Choe, Mi-Sun;Jin, Ung;Moon, Seok-Whan;Kim, Yong-Han;Kim, Chi-Kyung;Jo, Keon-Hyon;Kweon, Jong-Bum
    • Journal of Chest Surgery
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    • v.41 no.6
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    • pp.687-694
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    • 2008
  • Background: Although aortic valve sclerosis causes no significant hemodynamic alterations, it is associated with an increased risk of cardiovascular death and myocardial infarction. However, the role of ${\beta}_3$ integrin in aortic valve sclerosis remains unclear. Material and Method: Twenty male New Zealand rabbits were divided into two groups. Group 1 rabbits (n=10) received a normal chow diet, while group 2 (n=10) rabbits received a diet containing 1% cholesterol for 12 weeks. After the rabbits were euthanized, their aortic valves and ascending aortas were excised for analysis. Result: Total serum cholesterol ($2,148.3{\pm}1,012.5\;mg/dL$ versus $53.7{\pm}31.8\;mg/dL$, p<0.05), triglyceride ($240.4{\pm}218.3\;mg/dL$ versus $31.6{\pm}6.4\;mg/dL$, p<0.05), and low density lipoprotein (LDL)-cholesterol($2,065.3{\pm}960.9\;mg/dL$ versus $29.1{\pm}30.9\;mg/dL$, p<0.05) levels were significantly higher in the cholesterol diet group compared with the normal diet group. Myofibroblasts and macrophages were more highly expressed in the aortic valve leaflets of rabbits in the cholesterol diet group than of those in the normal diet group. A real-time polymerase chain reaction revealed decreased ${\beta}_3$ integrin mRNA levels in the hypercholesterolemic aortic valves and aortas. Conclusion: The present study shows that hypercholesterolemia induces aortic valve sclerosis. These findings suggest that alterations in ${\beta}_3$ integrin may playa role in the development of aortic valve sclerosis.

Size-dependent Transcriptional Modulation of Genes Involved in Cytochrome P450 Family in the Brackish Water Flea Diaphanosoma celebensis Exposed to Polystyrene Beads (기수산물벼룩 Diaphanosoma celebensis의 미세플라스틱 노출에 따른 크기 의존적 Cytochrome P450 유전자의 발현 양상)

  • Min Jeong Jeon;Je-Won Yoo;Young-Mi Lee
    • Journal of Marine Life Science
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    • v.8 no.2
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    • pp.104-114
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    • 2023
  • As plastic usage increases globally, the amount of plastic waste entering the marine environment is steadily rising. Microplastics, in particular, can be ingested by marine organisms and accumulated in their digestive tracts, causing harmful effects on their growth and reproduction. Cytochrome P450 (CYP) enzymes are known to metabolize various environmental pollutants as detoxification enzymes, but their role in crustaceans is not well understood. In this study, sequences of nine CYP genes (CYP370A4, CYP370C5 from clan 2; CYP350A1, CYP350C5, CYP361A1 from clan 3; CYP4AN-like, CYP4AP2, CYP4AP3, CYP4C33-like1 from clan 4) were analyzed using conserved domains in the brackish water flea Diaphanosoma celebensis. Additionally, after exposure to three different sizes of polystyrene beads (0.05-, 0.5-, 6-㎛ PS beads; 0.1, 1, and 10 mg/L) for 48 hours, the expression of these nine CYP genes were investigated using real-time reverse transcription polymerase chain reaction (RT-PCR). The results showed that all CYP genes possessed conserved motifs, indicating that D. celebensis CYP has evolutionarily conserved functions. Among these CYP genes, the expression of CYP370C5, CYP360A1, and CYP4C122 showed a significant increase after exposure to 0.05-㎛ PS beads, suggesting their involvement in PS metabolism. This research will contribute to understanding the molecular mode of actions of microplastics on marine invertebrates.

Comparison of PANA RealTyper HPV Kit with AdvanSure HPV GenoBlot Assay for Human Papillomavirus Genotyping (인유두종바이러스 유전자형 검사법 PANA RealTyper HPV Kit와 AdvanSure HPV GenoBlot Assay의 비교)

  • Kim, Yi Hyeon;Chung, Hae-Sun;Lee, Miae
    • Annals of Clinical Microbiology
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    • v.21 no.4
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    • pp.86-91
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    • 2018
  • Background: The PANA RealTyper HPV kit (PANAGENE, Korea; PANA RealTyper) was developed to genotype human papillomavirus (HPV) and was based on multiplex real-time PCR amplification and melting curve analysis. In this study, we compared PANA RealTyper to the AdvanSure HPV GenoBlot assay (LG Life Sciences, Korea; AdvanSure assay) and attempted to evaluate the performance of PANA RealTyper. Methods: A total of 60 cervical specimens were collected from women undergoing routine cervical cancer screening. The AdvanSure assay and PANA RealTyper kit identified the same 20 high-risk genotypes. However, the AdvanSure assay identified 15 low-risk genotypes, while the PANA RealTyper kit identified only 2 but detected 18 low-risk genotypes. Results: Among the total 60 specimens, 54 high-risk genotypes (40 specimens) and 20 low-risk genotypes (18 specimens) were detected. The agreement rates of the assays ranged from 94.4 to 100% for high-risk genotypes. Among 9 genotypes that were positive in the PANA RealTyper kit but negative in the AdvanSure assay, 7 were confirmed as true positive (HPV genotypes 16 (n=1), 39 (n=1), 52 (n=1), 58 (n=2), 68 (n=2)). Among 4 genotypes that were negative in the PANA RealTyper kit but positive in the AdvanSure assay, 3 were confirmed as HPV genotype 59. Among the 19 low-risk genotypes positive in the AdvanSure assay, there were 2 cases of HPV 6 and 1 case of HPV 11. In comparison, only 1 positive case of HPV 6 was determined by the PANA RealTyper kit. Conclusion: The PANA RealTyper kit was comparable with the AdvanSure assay. The PANA RealTyper kit would be useful and suitable for HPV genotyping in the clinical laboratory.

miR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells

  • Ning Song;Jun Luo;Lian Huang;Xiaoying Chen;Huimin Niu;Lu Zhu
    • Animal Bioscience
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    • v.36 no.10
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    • pp.1488-1498
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    • 2023
  • Objective: αS1-Casein is more closely associated with milk allergic reaction than other milk protein components. microRNA (miRNA) is a class of small non-coding RNAs that modulate multiple biological progresses by the target gene. However, the post-transcriptional regulation of αS1-casein expression by miRNA in ruminants remains unclear. This study aims to explore the regulatory roles of miR-380-3p on αS1-casein synthesis in goat mammary epithelial cells (GMEC). Methods: αS1-Casein gene and miR-380-3p expression was measured in dairy goat mammary gland by quantitative real-time polymerase chain reaction (qRT-PCR). miR-380-3p overexpression and knockdown were performed by miR-380-3p mimic or inhibitor in GMEC. The effect of miR-380-3p on αS1-casein synthesis was detected by qRT-PCR, western blot, luciferase and chromatin immunoprecipitation assays in GMEC. Results: Compared with middle-lactation period, αS1-casein gene expression is increased, while miR-380-3p expression is decreased during peak-lactation of dairy goats. miR-380-3p reduces αS1-casein abundance by targeting the 3'-untranslated region (3'UTR) of αS1-casein mRNA in GMEC. miR-380-3p enhances β-casein expression and signal transducer and activator of transcription 5a (STAT5a) activity. Moreover, miR-380-3p promotes β-casein abundance through target gene αS1-casein, and activates β-casein transcription by enhancing the binding of STAT5 to β-casein gene promoter region. Conclusion: miR-380-3p decreases αS1-casein expression and increases β-casein expression by targeting αS1-casein in GMEC, which supplies a novel strategy for reducing milk allergic potential and building up milk quality in ruminants.

The Analysis of Expression of Autoinducer Synthesis Genes Involved in Quorum Sensing among Catheter Associated Bacteria (요로감염에 관여하는 카테터 내 박테리아의 Quorum Sensing 관련 autoinducer 합성 유전자의 발현분석)

  • Lee, Mi-Hye;Seo, Pil-Soo;Lee, Ji-Youl;Peck, Kyong-Ran;Lee, Sang-Seob
    • Korean Journal of Microbiology
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    • v.42 no.4
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    • pp.277-285
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    • 2006
  • The most biofilm forming bacteria in catheter, Esctherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mRNA expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mRNA expression oil single strains culture were shown at $2.4{\times}10^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $6.9{\times}10^4$ CFU/ml of P. aeruginosa for rhlI and lasI. Also, in mixed culture of three, initial cell densities of mRNA expression were appear to at $7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $2.1{\times}10^5$ CFU/ml of P. aeruginosa for rhlI and lasI. Each AIs synthetic gene was expressed in initial cell density and the mRNA expression of the genes were detected continously during 30 days. And then, the quantification of mRNA expression level of ygaG, rhlI, last, and luxS which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mRNA levels of ygaG, rhlI, lasI, and luxS from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygaG, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa rhlI and lasI, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of S. aureus luxS, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mRNA expression of rhlI and lasI of P. aeruginosa showed the highest efficency among four AIs synthetic genes.

Prognostic Significance of CD44v6/v7 in Acute Promyelocytic Leukemia

  • Chen, Ping;Huang, Hui-Fang;Lu, Rong;Wu, Yong;Chen, Yuan-Zhong
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.8
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    • pp.3791-3794
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    • 2012
  • CD44v, especially splice variants containing exon v6, has been shown to be related closely to development of different tumors. High levels of CD44v6/v7 have been reported to be associated with invasiveness and metastasis of many malignancies. The objective of this study was to detect expression of CD44v6-containing variants in patients with acute promyelocytic leukemia (APL) and evaluate the potential of CD44v6/v7 for risk stratification. Reverse transcription polymerase chain reaction (RT-PCR) followed by PCR product purification, ligation into T vectors and positive clone sequencing were used to detect CD44 v6-containing variant isoforms in 23 APL patients. Real-time quantitative PCR of the CD44v6/v7 gene was performed in patients with APL and in NB4 cells that were treated with all-trans retinoic acid (ATRA) or arsenic trioxide ($As_2O_3$). Sequencing results identified four isoforms (CD44v6/v7, CD44v6/v8/v10, CD44v6/v8/v9/v10, and CD44v6/v7/v8/v9/v10) in bone marrow mononuclear cells of 23 patients with APL. The level of CD44v6/v7 in high-risk cases was significantly higher than those with low-risk. Higher levels of CD44v6/v7 were found in three patients with central nervous system relapse than in other patients inthe same risk group. Furthermore, in contrast to ATRA, only $As_2O_3$ could significantly down-regulate CD44v6/v7 expression in NB4 cells. Our data suggest that CD44v6/v7 expression may be a prognostic indicator for APL.

Impact of phosphorus application on the indigenous arbuscular mycorrhizal fungi, soybean growth and yield in a 5-year phosphorus-unfertilized crop rotation

  • Higo, Masao;Sato, Ryohei;Serizawa, Ayu;Gunji, Kento;Suzuki, Daisuke;Isobe, Katsunori
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2017.06a
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    • pp.351-351
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    • 2017
  • Arbuscular mycorrhizal fungi (AMF) are particular soil fungi that benefit many crops and require a symbiosis with plant roots to survive. In our previous study, there was a positive correlation between AMF root colonization and soybean grain yield in a four-year consecutive winter cover crop-soybean rotational system without phosphorus fertilizer. It is suggested that higher AMF root colonization can be a better solution for improving soybean growth and grain yield in P-limited soil. Our purpose in this study was to test the hypothesis that a P application is the main factor improving soybean growth, P nutrition and grain yield, and the benefit from AMF to soybean P uptake and growth in a P-limited soil. Impact of a P application on AMF root colonization and communities in soybean roots and their potential contribution to soybean growth and P nutrition under a five-year P-unfertilized crop rotational system were investigated over two-years. In this study, four cover crop treatments included 1) wheat (Triticum aestivum); 2) red clover (Trifolium pratense); 3) rapeseed (Brassica napus); and 4) fallow in the crop rotation. The amount of triple superphosphate as a P fertilizer applied rate after cultivation of cover crops was 120 and $360k\;ha^{-1}$ in 2014 and 2015, respectively. Soybean roots were sampled at full-flowering and analyzed for AMF communities using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time PCR (qPCR) techniques. The AMF root colonization in the soybean roots at full bloom stage was significantly influenced by cover crop and P application throughout the two-year rotation. The two-year rotation of different cover crops or fallow impacted the molecular diversity of AMF communities colonizing roots of soybean. Redundancy analysis (RDA) indicated that AMF communities colonizing roots of soybean were significantly different among cover crop rotations. The AMF communities colonizing roots of soybean were clearly influenced by a P application in the two-year trial. Moreover, a P application may have positively impacts on the AMF communities under P-deficit soil due to the continuous cover crop-soybean rotational system without a P fertilizer.

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Molecular Cloning of the cDNA of Heat Shock Protein 88 Gene from the Entomopathogenic Fungus, Paecilomyces tenuipes Jocheon-1

  • Liu, Ya-Qi;Park, Nam Sook;Kim, Yong Gyun;Kim, Keun Ki;Park, Hyun Chul;Son, Hong Joo;Hong, Chang Ho;Lee, Sang Mong
    • International Journal of Industrial Entomology and Biomaterials
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    • v.28 no.2
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    • pp.71-84
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    • 2014
  • The full-length heat shock protein 88 (HSP88) complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening the Paecilomyces tenuipes (P. tenuipes) Jocheon-1 Uni-Zap cDNA library and performing 5' RACE polymerase chain reaction (PCR). The P. tenuipes Jocheon-1 HSP88 cDNA contained an open reading frame (ORF) of 2,139-basepair encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipe s Jocheon-1 HSP88 cDNA showed 77% identity to Nectria haematococca HSP88 and 45-76% identity to other fungal homologous HSP88s. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade. The P. tenuipes Jocheon-1 HSP88 also contained the conserved ATPase domain at the N-terminal region. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88 kilodalton (kDa) polypeptide in baculovirus-infected insect Sf9 cells. Under higher temperature conditions for the growth of the entomopathogenic fungus, mRNA expression of P. tenuipes Jocheon-1 HSP88 was quantified by real time PCR (qPCR). The results showed that heat shock stress induced a higher level of mRNA expression compared to normal growth conditions.

Spatio-temporal distributions of the newly described mixotrophic dinoflagellate Yihiella yeosuensis (Suessiaceae) in Korean coastal waters and its grazing impact on prey populations

  • Jang, Se Hyeon;Jeong, Hae Jin
    • ALGAE
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    • v.35 no.1
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    • pp.45-59
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    • 2020
  • To investigate the spatio-temporal distributions of the mixotrophic dinoflagellate Yihiella yeosuensis in Korean coastal waters and its grazing impact on prey populations, water samples were seasonally collected from 28 stations in the East, West, and South Seas of Korea and Jeju Island from April 2015 to October 2018. The abundances of Y. yeosuensis in the water samples were quantified using quantitative real-time polymerase chain reaction (qPCR). Simultaneously, the physical and chemical properties of water from all sampled stations were determined, and the abundances of the optimal prey species of Y. yeosuensis, the prasinophyte Pyramimonas sp. and the cryptophyte Teleaulax amphioxeia, were quantified using qPCR. Y. yeosuensis has a wide distribution, as is reflected by the detection of Y. yeosuensis cells at 23 sampling stations; however, this distribution has a strong seasonality, which is indicated by its detection at 22 stations in summer but only one station in winter. The abundance of Y. yeosuensis was significantly and positively correlated with those of Pyramimonas sp. and T. amphioxeia, as well as with water temperature. The highest abundance of Y. yeosuensis was 48.5 cells mL-1 in Buan in July 2017, when the abundances of Pyramimonas sp. and T. amphioxeia were 917.6 and 210.4 cells mL-1, respectively. The growth rate of Y. yeosuensis on Pyramimonas sp., calculated by interpolating the growth rates at the same abundance, was 0.49 d-1, which is 37% of the maximum growth rate of Y. yeosuensis on Pyramimonas sp. obtained in the laboratory. Therefore, the field abundance of Pyramimonas sp. obtained in the present study can support a moderate positive growth of Y. yeosuensis. The maximum grazing coefficient for Y. yeosuensis on the co-occurring Pyramimonas sp. was 0.42 d-1, indicating that 35% of the Pyramimonas sp. population were consumed in 1 d. Therefore, the spatio-temporal distribution of Y. yeosuensis in Korean coastal waters may be affected by those of the optimal prey species and water temperature. Moreover, Y. yeosuensis may potentially have considerable grazing impacts on populations of Pyramimonas sp.

Clinical and Laboratory Finding of the 2009 Pandemic influenza A (H1N1) in Children (소아에서 2009 신종 인플루엔자 A (H1N1) 바이러스 감염의 임상적 특징)

  • Sohn, Yu Rak;Park, Su Hyun;Kim, Won Duck
    • Pediatric Infection and Vaccine
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    • v.18 no.2
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    • pp.173-181
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    • 2011
  • Purpose : 2009 Pandemic influenza A (H1N1) virus was identified in March 2009 and subsequently caused worldwide outbreaks. We described the clinical and epidemiological characteristics of H1N1 influenza infection. Methods : We used retrospective medical chart reviews to collect data on the visiting patients from a single institute. H1N1 infection was confirmed in specimens with the use of a RT-PCR (real time reverse transcriptase polymerase chain reaction assay). Result : 6,836 patients had H1N1 RT-PCR test, and 2,781 were confirmed with H1N1 virus infection. 158 patients (5.7%) had hospital treatment and inpatients were significantly younger (5.4${\pm}$3.3 years) than outpatients (7.5${\pm}$3.9 years) among H1N1 virus confirmed patients. Oxygen, steroid, immunoglobulin, ventilator treatment was provided in a substantial proportion among pneumonia patients accompanying wheezy respiration. In addition more intensive care was needed in patients accompanying segmental, lobar, interstitial, mixed pneumonia and lung effusion (27.2%) than patients with bronchopneumonia (7.3%) among H1N1 virus infection confirmed patients. Seventy-one infants had oseltamivir treatment out of 83 infants under 1 year, and no significant side effects and complications were identified. Conclusion : In 2009 pandemic influenza A (H1N1), hospital treatment was needed in younger patients. Early intensive care was needed in pneumonia patients accompanying wheezy respiration, and patients accompanying segmental, lobar, interstitial, mixed pneumonia and lung effusion.