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

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Rehmannia Glutinosa Pharmacopuncture Solution Regulates Functional Activation, FcεRI Expression, and Signaling Events in Mast Cells

  • Kang, Kyung-Hwa;Lee, Kyung-Hee;Yoon, Hyun-Min;Jang, Kyung-Jeon;Song, Chun-Ho;Kim, Cheol-Hong
    • Journal of Pharmacopuncture
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    • v.15 no.4
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    • pp.32-41
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    • 2012
  • Objectives: Rehmannia glutinosa pharmacopuncture solution (RGPS) was investigated to determine both its anti-allergic inflammatory effects on mast cells and its detailed mechanism of actions. Methods: We investigated whether RGPS suppress cytokines, enzymes, $Fc{\varepsilon}RI$ expression and $Fc{\varepsilon}RI$-mediated signaling in RBL-2H3 cells stimulated with anti-DNP IgE/DNP-HSA. The suppressive effects of RGPS on the levels of cytokines such as IL-$1{\beta}$, IL-6 and GM-CSF were measured using emzyme-linked immunospecific assay (ELISA). The mRNA expression levels of cytokines, enzymes (HDC2, COX-1, COX-2 and 5LO) and $Fc{\varepsilon}RI$ ${\alpha}{\beta}{\gamma}$ subunits were measured using reverse transcription polymerase chain reaction (RT-PCR) method. The activation of $Fc{\varepsilon}RI$-mediated signaling was examined using Western blot analyses. Results: RGPS suppressed production of proinflammatory cytokines (IL-$1{\beta}$, IL-6, and GM-CSF) in stimulated RBL-2H3 cells significantly (p < 0.05). RGPS also suppressed mRNA expression of inflammatory enzymes (HDC2, COX-1, COX-2, 5LO). In addition, mRNA expression levels of $Fc{\varepsilon}RI{\alpha}$, $Fc{\varepsilon}RI{\beta}$and $Fc{\varepsilon}RI{\gamma}$ were lowered by treatment with RGPS. Finally, RGPS prevented phosphrylation of Lyn, Syk, LAT, Gab2, PLC ${\gamma}1/2$, PI3K, Akt, cPLA2 and $I{\kappa}B{\alpha}$. Conclusions: RGPS effectively suppresses mast cell activations such as degranulation and inflammatory response via down-regulation of the $Fc{\varepsilon}RI$-mediated signaling pathways in IgE/Ag-stimulated mast cells.

Comparison of Virulence Factors, Phylogenetic Groups and Ciprofloxacin Susceptibility of Escherichia coli Isolated from Healthy Students and Patients with Urinary Tract Infections in Korea

  • Park, Min;Park, Soon-Deok;Kim, Sa-Hyun;Woo, Hyun-Jun;Lee, Gyu-Sang;Kim, Hyun-Woo;Yang, Ji-Young;Cho, Eun-Hee;Uh, Young;Kim, Jong-Bae
    • Biomedical Science Letters
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    • v.18 no.2
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    • pp.146-151
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    • 2012
  • Urinary tract infection (UTI) is one of the most common bacterial infections and is predominantly caused by uropathogenic Escherichia coli (UPEC). UPEC strains generally possess several genes encoding virulent factors, which are mostly adhesins, toxins, bacteriocin and siderophores. E. coli is composed of four main phylogenetic group (A, B1, B2, D) and virulent extra-intestinal strains mainly belong to groups B2 and D. Prescription of ciprofloxacin, a kind of fluoroquinolone group antibiotics, is increasing now a days, but resistance to this drug is also increasing. A total of 188 strains of E. coli were collected. Thirteen strains were collected from healthy students in 2011 and 175 strains from patients with urinary tract infection in 2010. Virulence factor genes (papC, fimG/H, sfaD/E, hlyA, cnf1, and usp) were amplified by polymerase chain reaction (PCR) methods for phylogenetic group (A, B1, B2, D) detection. Ciprofloxacin susceptibility test was performed by disk diffusion method. The identified virulence factors (VFs), phylogenetic groups and ciprofloxacin resistance in 13 E. coli strains isolated from healthy students were papC (15.4%), fimG/H (76.9%), sfaD/E (30.8%), hlyA (23.1%), cnf1 (23.1%), usp (7.7%), phylogenetic group A (23%), B1 (8%), B2 (46%), D (23%) and ciprofloxacin resistance (7.7%), while those of in 175 E. coli strains isolated from patients with UTI were papC (41.1%), fimG/H (92.5%), sfaD/E (30.3%), hlyA (10.3%), cnf1 (30.3%), usp (27.4%), phylogenetic group A (9.1%), B1 (5.1%), B2 (60.6%), D (25.1%) and ciprofloxacin resistance (29.7%). In this study, 10 out of 13 E. coli strains (76.9%) from healthy students were found to possess more than one virulence factor associated with adhesion. In addition, one E. coli strain isolated from healthy students who had never been infected with UPEC showed ciprofloxacin resistance. According to these results between the virulence factors and phylogenetic groups it was closely associated, and UPEC strains isolated from patients showed high level of ciprofloxacin resistance.

Incidence of Viral Diseases on Red Pepper in Yeongyang-Gun, Gyeongbuk Provice (경북 영양지역 노지고추의 바이러스병 발생양상)

  • Kwon, Oh-Hun;Lee, Joong-Hwan;Jang, Kil-Su;Kim, Dong-Jin;Kim, Chan-Yong;Jeon, Su-Gyeong;Kwon, Jung-Bae;Jung, Hee-Young
    • Research in Plant Disease
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    • v.23 no.3
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    • pp.234-240
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    • 2017
  • Incidence of virus diseases in red pepper of open field in Yeongyang-Gun, Gyeongbuk Province was investigated by reverse transcription-polymerase chain reaction in 2012-2016. The infection rate of Cucumber mosaic virus (CMV), Broad bean wilt virus 2 (BBWV2), Pepper mottle virus, Potato virus Y, Pepper mild mottle virus and Tomato spotted wilt virus was 46.1%, 41.5%, 2.0%, 2.0%, 4.4% and 0.1% respectively. Incidence rate of single and mixed infection was 31.2% and 62.6%. Most of single infections were CMV and BBWV2. Among mixed infections, the incidence rate of CMV+BBWV2 mixed infection was the highest as 49.3% and most of mixed infections of triplex and tetraplex included CMV+BBWV2 mixed infection. CMV single infection caused mosaic, chlorosis, yellowing and vein necrosis and BBWV2 single infection induced cholosis and mosaic. CMV+BBWV2 mixed infection caused severe mosaic with chlorosis or malformation.

사료내 감태 및 감태로부터 추출한 crude lectin의 첨가가 육계의 생산성 및 면역반응에 미치는 영향

  • 김성권;유선종;안병기;박근규;이훈택;송창선;허억;강창원
    • Proceedings of the Korea Society of Poultry Science Conference
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    • 2004.11a
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    • pp.23-25
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    • 2004
  • This study was conducted to investigate the effects of dietary supplementation of Ecklonia cava kjellman(ECK) and crude lectin extracted from ECK (CLEEC) on performances and immune responses in broiler chicks. A total of two hundreds thirty four 1 day old male broiler chicks (Ross) were fed corn-soy based diets containing 0 % (with or without vaccination and Salmonella challenge), 1.0 % ECK, 0.05 %, 0.1 % and 0.3 % CLEEC for 38 days and vaccinated against inactivated ND-IB combined oil vaccine on the fourth day. After S. gallinarum challenge. mortality was measured daily. The spleens of birds were removed for RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) with primer sets for IFN-ν, IL-2, IL-6 and ${\beta}$-actin were performed with RNA samples. At the 28th day, pancreas weights were heavier 0.3 % CLEEC than 1.0 % ECK group. At the 21st day after ND-IB oil vaccine injection, dietary supplementation of ECK and CLEEC tended to increase or significantly (P<0.05) improved ND or IB titer compared to the positive control. Mortality was significantly (P<0.05) decreased by dietary CLEEC treatments. Chicken splenic IFN-ν, IL-2, and IL-6 cytokines mRNA expressions were enhanced by challenge with S, gallinarum. Dietary treatments did not affect mRNA expression of IFN-ν. However, IL-2 and IL-6 expressions in Salmonella challenged birds that fed the 1.0 % ECK or 0.05 % CLEEC groups were enhanced (P<0.05) compare to the positive control. The results demonstrated that dietary ECK and CLEEC enhanced humoral and cellular immunity and therefore. it can be concluded that dietary supplementation of ECK and CLEEC can be used as a feed additive for enhancement of immunocompetence without any adverse effects in broiler chicks.

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Genetic analyses of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea

  • Yu, Hak-Sun;Choi, Kyung-Hee;Kim, Hyo-Kyung;Kong, Hyun-Hee;Chung, Dong-Il
    • Parasites, Hosts and Diseases
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    • v.39 no.2
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    • pp.161-170
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    • 2001
  • We conducted both the small subunit ribosomal DNA (SSU rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and mitochondrial (mt) DNA RFLP analyses for a genetic characterization of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Twenty-three strains of Acanthamoeba from the American Type Culture Collection and twelve clinical isolates from Korean patients were used as reference strains. Thirty-nine isolates from contact lens storage cases were classified into seven types (KA/LS1 , KA/LS2, KA/LS4, KA/LS5, KA/LS7 KA/LS18, KA/LS31). Four types (KA/LS1 , KA/LS2, KA/LS5, KA/LS18) including 33 isolates were regarded as A. castellanii complex by riboprints. KA/LS1 type was the most predominant (51.3%) in the present survey area, followed by KA/LS2 (20.9%), and KA/LSS (7.7%) types. Amoebae of KA/LS1 type had the same mtDNA RFLP and riboprint patterns as KA/E2 and KA/E12 strains, clinical isolates from Korean keratitis patients. Amoebae of KA/LS2 type had the identical mtDNA RFLP patterns with A. castellanii Ma strain, a corneal isolate from an American patient as amoebae of KA/LS5 type, with KA/E3 and KA/E8 strains from other Korean keratitis patients. Amoebae of KA/LS 18 type had identical patterns with JAC/E1, an ocular isolate from a Japanese patient. Three types , which remain unidentified at species level, were not corresponded with any clinical isolate in their mtDNA RFLP and riboprint patterns. Out of 39 isolates analyzed in this study, mtDNA RFLP and riboprint patterns of 33 isolates (84.6%) were identical to already known clinical isolates, and therefore, they may be regarded as potentially keratopathogenic. These results suggest that contact lens wearers in Seoul should pay more attention to hygienic maintenance of contact lens storage cases for the prevention of Acanthamoeba keratitis.

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Enhanced Expression of TREK-1 Is Related with Chronic Constriction Injury of Neuropathic Pain Mouse Model in Dorsal Root Ganglion

  • Han, Hyo Jo;Lee, Seung Wook;Kim, Gyu-Tae;Kim, Eun-Jin;Kwon, Byeonghun;Kang, Dawon;Kim, Hyun Jeong;Seo, Kwang-Suk
    • Biomolecules & Therapeutics
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    • v.24 no.3
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    • pp.252-259
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    • 2016
  • Neuropathic pain is a complex state showing increased pain response with dysfunctional inhibitory neurotransmission. The TREK family, one of the two pore domain $K^+$ (K2P) channel subgroups were focused among various mechanisms of neuropathic pain. These channels influence neuronal excitability and are thought to be related in mechano/thermosensation. However, only a little is known about the expression and role of TREK-1 and TREK-2, in neuropathic pain. It is performed to know whether TREK-1 and/or 2 are positively related in dorsal root ganglion (DRG) of a mouse neuropathic pain model, the chronic constriction injury (CCI) model. Following this purpose, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot analyses were performed using mouse DRG of CCI model and compared to the sham surgery group. Immunofluorescence staining of isolectin-B4 (IB4) and TREK were performed. Electrophysiological recordings of single channel currents were analyzed to obtain the information about the channel. Interactions with known TREK activators were tested to confirm the expression. While both TREK-1 and TREK-2 mRNA were significantly overexpressed in DRG of CCI mice, only TREK-1 showed significant increase (~9 fold) in western blot analysis. The TREK-1-like channel recorded in DRG neurons of the CCI mouse showed similar current-voltage relationship and conductance to TREK-1. It was easily activated by low pH solution (pH 6.3), negative pressure, and riluzole. Immunofluorescence images showed the expression of TREK-1 was stronger compared to TREK-2 on IB4 positive neurons. These results suggest that modulation of the TREK-1 channel may have beneficial analgesic effects in neuropathic pain patients.

Genetic Polymorphisms of UGT1A and their Association with Clinical Factors in Healthy Koreans

  • Kim, Jeong-Oh;Shin, Jeong-Young;Lee, Myung-Ah;Chae, Hyun-Suk;Lee, Chul-Ho;Roh, Jae-Sook;Jin, Sun-Kyung;Kang, Tae-Sun;Choi, Jung-Ran;Kang, Jin-Hyoung
    • Genomics & Informatics
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    • v.5 no.4
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    • pp.161-167
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    • 2007
  • Glucuronidation by the uridine diphosphateglucuronosy-ltransferase 1A enzymes (UGT1As) is a major pathway for elimination of particular drugs and endogenous substances, such as bilirubin. We examined the relation of eight single nucleotide polymorphisms (SNPs) and haplotypes of the UGT1A gene with their clinical factors. For association analysis, we genotyped the variants by direct sequencing analysis and polymerase chain reaction (PCR) in 218 healthy Koreans. The frequency of UGT1A1 polymorphisms, -3279T>G, -3156G>A, -53 $(TA)_{6>7}$, 211G>A, and 686C>A, was 0.26, 0.12, 0.08, 0.15, and 0.01, respectively. The frequency of -118 $(T)_{9>10}$ of UGT1A9 was 0.62, which was significantly higher than that in Caucasians (0.39). Neither the -2152C>T nor the -275T>A polymorphism was observed in Koreans or other Asians in comparison with Caucasians. The -3156G>A and -53 $(TA)_{6>7}$ polymorphisms of UGT1A were significantly associated with platelet count and total bilirubin level (p=0.01, p=0.01, respectively). Additionally, total bilirubin level was positively correlated with occurrence of the UGT1A9-118 $(T)_{9>10}$ rare variant. Common haplotypes encompassing six UGT1A polymorphisms were significantly associated with total bilirubin level (p=0.01). Taken together, we suggest that determination of the UGT1A1 and UGT1A9 genotypes is clinically useful for predicting the efficacy and serious toxicities of particular drugs requiring glucuronidation.

The Study on the Sexual Difference in the Cause and the Time of Casualty and in the Size of the Fairy Pitta (Pitta nympha) through DNA Analysis in Republic of Korea (DNA 분석에 의한 팔색조의 암수 구분 및 암수별 피해 현황 그리고 크기 차이에 관한 연구)

  • Kim, Eun-Mi;Jeon, Yeon-Seon;Jeong, Gil-Sang;Kim, Se-Jae;Kang, Chang-Wan;Oh, Mi-Rea;Noh, Pu-Reum;Won, Hyun-Kyu
    • Journal of Environmental Science International
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    • v.23 no.8
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    • pp.1447-1453
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    • 2014
  • The differentiation of sex is important for species preservation. However, Fairy Pitta is sexually monomorphic and sex of an individual is indistinguishable with its external characteristics. We determined the sex of Fairy Pitta through DNA analysis and investigated the causes and time of injury and mortality and the size based on sex. We collected 21 samples at Jeju Island, Korean Peninsula from 2004 to 2013 and extracted DNA from them and amplified chromo helicase DNA-binding gene from Z and W chromosomes through Polymerase Chain Reaction (PCR). We confirmed their sex with the banding pattern through Agarose gel electrophoresis, i.e. male (ZZ): one banded and female (ZW) two banded. We distinguished the sex of 17 of 21 samples resulting in 9 males and 8 females. Most casualties were recorded in adult of both sexes. Causes of injury and mortality proved that female casualties occurred from window strikes, dehydration, car accident, predation by natural enemies, and male occurred from window strikes, car accident and dehydration. The time of injury and mortality in adults differ by sex. There was no difference between sexes in any of the six size parameters. As the time of injury and mortality differ by sex, the survey on the role and ecological nature by sex in breeding season must be carried out in the future. External measurements may not be reliable for sexing of Fairy Pitta and other traits such as vocal or characteristics are required to identify the sex of individuals in the field.

Anti-inflammatory Effects of Wiryeong-tang Mix Soft Extract (위령탕 혼합단미연조엑스의 항염증 효과)

  • Kim, Myoung-Jin;Kim, Se-Jin;Nam, Won-Hee;Leem, Hyun-Hee;Jeon, Young-Hee;Son, Su-Mi;Kim, Jung-Ok
    • Herbal Formula Science
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    • v.28 no.4
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    • pp.339-349
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    • 2020
  • Objectives : Wiryeong-tang (WRT) is a traditional herbal medicine used to treat kidney-related diseases. However, the anti-inflammatory and anti-gastritis effect of Wiryeong-tang was not well known. Therefore, we experimented to confirmed the anti-inflammatory and anti-gastritis effects of Wiryeong-tang. Methods : The RAW 264.7 cells were pre treated with Wiryeong-tang mix soft extract (WRT-mse; 50, 100 and 200 ㎍/mL) for 1 hrs, and then incubated with lipopolysaccharide (LPS; 500 ng/mL). Cell viability was measured by the MTT method, and nitric oxide (NO) was measured with griess reagent. In addition, pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). For anti-gastritis effect in vivo, acute gastritis was induced using 150 mM HCl/60% ethanol used ICR mice. WRT-mse (133 mg/kg) was pre treated for 3 days and then treated with 150 mM HCl/60% ethanol 1 hrs later. Then gastritis was observed and inflammatory cytokines in the gastric tissue was measured. Results : The 8 marker components of the WRT-mse were determined by simultaneous analysis using HPLC. WRT-mse was not toxic and inhibited pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α at NO production, protein and mRNA levels. Also, it was confirmed that WRT-mse improved bleeding and edema in gastritis, and suppresses inflammatory cytokines. Conclusion : In summary, our results suggest that the treatment of the WRT-mse reduced and improved the 150 mM HCl/60% ethanol induced acute gastritis and the inflammation caused by LPS stimulation in RAW 264.7 cells. Therefore, this study may provide useful drug or clinical evidence for WRT-mse to prevent inflammation.

Pneumococcal Surface Protein A of Streptococcus pneumoniae Isolates from Koreans (한국인에서 Streptococcus pneumoniae 분리주의 폐구균 표면 단백 A)

  • Kim, Kyung Hyo
    • Clinical and Experimental Pediatrics
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    • v.48 no.11
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    • pp.1206-1211
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    • 2005
  • Purpose : Pneumococcal protein vaccine based on pneumococcal surface protein A (PspA) is in development with the potential to offer a broad range of protection against different strains. PspA elicits protection in mice against fatal sepsis as well as carriage and lung infection. This study was performed to investigate the frequency of PspA families among Streptococcus pneumoniae recovered from Korean children and adults. Methods : A total of 89 pneumococcal isolates was included in the study. They were capsule serotyped by the slide agglutination assay with commercial antisera. PspA families were determined with polymerase chain reaction using the pair of primers for family 1 and family 2. Results : Seventeen pneumococcal serotypes were found in a total of 89 isolates. PspA typing was able to ascertain 79 of the 89 isolates (88.8 percent). Among these, 20 (22.5 percent) isolates were family 1 PspA, 59 (66.3 percent) were family 2. Moreover, because 9 (10.1 percent) isolates were of positive reactions for both, families 1 and 2 primers, the potential coverage of PspA vaccine was 98.9 percent. PspA families were not associated with age group, source of isolates, or penicillin susceptibility. However, the relative distribution of family 1 isolates to family 2 isolates was significantly different over capsular serotypes. Conclusion : The finding that 98.9 percent of Korean isolates belonging to PspA families 1 and 2 support the hypothesis that a human PspA vaccine covering a few PspA families could be broadly effective. The monitoring of the PspA families derived from large population-based isolates will be necessary in the context of vaccine development.