• Title/Summary/Keyword: IS-PCR

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Selective Expansion of TCR $V{\beta}3$+CD4+T Cells in Collagen-induced Arthritis in DBA/1 Mice (콜라겐 유도 관절염에서 콜라겐 항원 특이 $V{\beta}3$+CD4+T 세포의 선택적 증식)

  • Lee, Jae-Seon;Cho, Mi-La;Lee, Jung-Eun;Min, So-Youn;Yoon, Chong-Hyeon;Kim, Wan-Uk;Min, Jun-Ki;Park, Sung-Hwan;Kim, Ho-Youn
    • IMMUNE NETWORK
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    • v.5 no.2
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    • pp.78-88
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    • 2005
  • Background: Collagen-induced arthritis (CIA) in mice is animal model of autoimmune disease known as rheumatic arthritis in human. We investigated CII-specific CD4+ T cell receptor usage in CIA mice. Methods: In CIA model, draining lymph node (dLN) CD4+ T cells and splenocytes at $3^{rd},\;5^{th},\;8^{th}$ week, we investigated CII-specific T cell proliferation, production of IL-17, IFN-${\gamma}$, TNF-${\alpha}$, IL-4 and IL-10. And we also performed anti-CII IgG Ab measurements in serum level, TCRV ${\beta}$ usage and T cell clonality with RT-PCR-SSCP analysis. Also, we performed proliferative response against CII when CII-specific T cell subset is deleted. Results: CIA mice showed more increase in the serum level of anti-CII IgG than normal mice after induction of arthritis. And the level of anti-CII IgG2a in CIA mice was increased after $3^{rd}$ week after primary immunization, while anti-CII IgG1 was decreased. Draining LN CD4+ T cells have proliferated against CII stimulation at $3^{rd}$ week after $1^{st}$immunization. CD4+T cells derived from dLN of CIA mice produced proinflammatory cytokine IFN-${\gamma}$, IL-17 etc. Draining LN CD4 T cells of CIA presented higher proportion of CD4+V ${\beta}3$+subset compared to those of normal mice at $3^{rd}$ week after $1^{st}$ immunization, and they were increased in proportion by CII stimulation. Draining LN CD4+ T cells without TCRV ${\beta}3+/V{\beta}8.1/8.2+/V{\beta}$10b+cells were not responsive against CII stimulation. But, CII-reactive response of TCRV ${\beta}3-/V{\beta}8.1/8.2-/V{\beta}$10b- T cells was recovered when $V{\beta}3+$ T cells were added in culture. Conclusion: Our results indicate that CD4+$V{\beta}3+$ T cells are selectively expanded in dLN of CIA mice, and their recovery upon CII re-stimulation in vitro, as well as the production Th1-type cytokines, may play pivotal role in CIA pathogenesis.

PERIPHERAL NERVE REGENERATION USING POLYGLYCOLIC ACID CONDUIT AND BRAIN-DERIVED NEUROTROPHIC FACTOR GENE TRANSFECTED SCHWANN CELLS IN RAT SCIATIC NERVE (BDNF 유전자 이입 슈반세포와 PGA 도관을 이용한 백서 좌골신경 재생에 관한 연구)

  • Choi, Won-Jae;Ahn, Kang-Min;Gao, En-Feng;Shin, Young-Min;Kim, Yoon-Tae;Hwang, Soon-Jeong;Kim, Nam-Yeol;Kim, Myung-Jin;Jo, Seung-Woo;Kim, Byung-Soo;Kim, Yun-Hee;Kim, Soung-Min;Lee, Jong-Ho
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.30 no.6
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    • pp.465-473
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    • 2004
  • Purpose : The essential triad for nerve regeneration is nerve conduit, supporting cell and neurotrophic factor. In order to improve the peripheral nerve regeneration, we used polyglycolic acid(PGA) tube and brain-derived neurotrophic factor(BDNF) gene transfected Schwann cells in sciatic nerve defects of SD rat. Materials and methods : Nerve conduits were made with PGA sheet and outer surface was coated with poly(lactic-co-glycolic acid) for mechanical strength and control the resorption rate. The diameter of conduit was 1.8mm and the length was 17mm Schwann cells were harvested from dorsal root ganglion(DRG) of SD rat aged 1 day. Schwann cells were cultured on the PGA sheet to test the biocompatibility adhesion of Schwann cell. Human BDNF gene was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into E1 deleted region of adenovirus shuttle vector, pAACCMVpARS. BDNF-adenovirus was multiplied in 293 cells and purified. The BDNF-Adenovirus was then infected to the cultured Schwann cells. Left sciatic nerve of SD rat (250g weighing) was exposed and 14mm defects were made. After bridging the defect with PGA conduit, culture medium(MEM), Schwann cells or BDNF-Adenovirus infected Schwann cells were injected into the lumen of conduit, respectively. 12 weeks after operation, gait analysis for sciatic function index, electrophysiology and histomorphometry was performed. Results : Cultured Schwann cells were well adhered to PGA sheet. Sciatic index of BDNF transfected group was $-53.66{\pm}13.43$ which was the best among three groups. The threshold of compound action potential was between 800 to $1000{\mu}A$ in experimental groups which is about 10 times higher than normal sciatic nerve. Conduction velocity and peak voltage of action potential of BDNF group was the highest among experimental groups. The myelin thickness and axonal density of BDNF group was significantly greater than the other groups. Conclusion : BDNF gene transfected Schwann cells could regenerate the sciatic nerve gap(14mm) of rat successfully.

Taurine exerts neuroprotective effects via anti-apoptosis in hypoxic-ischemic brain injury in neonatal rats (신생 흰쥐의 저산소성 허혈성 뇌손상에서 항세포사멸사를 통한 taurine의 신경보호 효과)

  • Jeong, Ji Eun;Kim, Tae Yeol;Park, Hye Jin;Lee, Kye Hyang;Lee, Kyung Hoon;Choi, Eun Jin;Kim, Jin Kyung;Chung, Hai Lee;Seo, Eok Su;Kim, Woo Taek
    • Clinical and Experimental Pediatrics
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    • v.52 no.12
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    • pp.1337-1347
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    • 2009
  • Purpose:Taurine (2-aminoethanesulfonic acid) is a simple sulfur-containing amino acid. It is abundantly present in tissues such as brain, retina, heart, and skeletal muscles. Current studies have demonstrated the neuroprotective effects of taurine, but limited data are available for such effects during neonatal period. The aim of this study was to determine whether taurine could reduce hypoxic-ischemic (HI) cerebral injury via anti-apoptosis mechanism. Methods:Embryonic cortical neurons isolated from Sprague-Dawley (SD) rats at 18 days gestation were cultured in vitro. The cells were divided into hypoxia group, taurine-treated group before hypoxic insult, and taurine-treated group after HI insult. In the in vivo model, left carotid artery ligation was performed in 7-day-old SD rat pups. The pups were exposed to hypoxia, administered an injection of 30 mg/kg of taurine, and killed at 1 day, 3 days, 1 week, 2 weeks, and 4 weeks after the hypoxic insult. We compared the expressions of Bcl-2, Bax, and caspase-3 among the 3 groups by using real- time polymerase chain reaction (PCR) and western blotting. Results:The cells in the taurine-treated group before hypoxic insult, although similar in appearance to those in the normoxia group, were lesser in number. In the taurine-treated group, Bcl-2 expression increased, whereas Bax and caspase-3 expressions reduced. Conclusion:Taurine exerts neuroprotective effects onperinatal HI brain injury due to its anti-apoptotic effect. The neuroprotective effect was maximal at 1-2 weeks after the hypoxic injury.

Genetic Variations of Chicken MC1R Gene and Associations with Feather Color of Korean Native Chicken (KNC) 'Woorimatdag' (토종 '우리맛닭' 부계 및 실용계에서 MC1R 유전자 변이 및 모색과의 연관성 분석)

  • Park, Mi Na;Kim, Tae-Hun;Lee, Hyun-Jeong;Choi, Jin Ae;Heo, Kang-Nyeong;Kim, Chong-Dae;Choo, Hyo-Jun;Han, Jae-Yong;Lee, Taeheon;Lee, Jun-Heon;Lee, Kyung-Tai
    • Korean Journal of Poultry Science
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    • v.40 no.2
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    • pp.139-145
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    • 2013
  • There are several loci controlling the feather color of birds, of which one of the most studied is Extended black (E) encoding the melanocortin 1-receptor (MC1R). Mutations in this gene affect the relative distribution of eumelanin, phaeomelanin. The association of feather color and sequence polymorphism in the melanocortin 1-receptor (MC1R) gene was investigated using Korean native chicken H breed (H_PL) and 'Woorimatdag' commercial chickens (Woorimatdag_CC). In order to correlate gene mutation to Korean native chicken feather color, single nucleotide polymorphism (SNP) from MC1R gene sequence were investigated. A total of 307 birds from H_PL and Woorimatdag_CC were used. H_PL have black, black-brown feather color and Woorimatdag_CC have black with brown spots or brown with black spots. There are 6 SNPs in MC1R gene, locus T69C, C212T, A274G, G376A, G636A, T637C. 3 SNPs are nonsynonymous that change amino acid. But it is difficult to find correlation of feather color and polymorphisms. It will be needed to increase the population of Korean native chicken H breed and correlation analysis of genetic variation with feather colors.

Clinical characteristics of severe respiratory syncytial virus infection requiring mechanical ventilation in neonatal period and early infancy (신생아기와 조기 영아기에 발생한 기계환기요법이 필요했던 중증 respiratory syncytial virus 감염의 임상적 특성)

  • Shin, Seung Han;Kim, Jae Ri;Lee, Jin-A;Choi, Chang Won;Kim, Ee-Kyung;Choi, Eun Hwa;Kim, Han-Suk;Kim, Beyong Il;Lee, Hoan Jong;Choi, Jung Hwan
    • Clinical and Experimental Pediatrics
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    • v.51 no.4
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    • pp.372-376
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    • 2008
  • Purpose : To identify clinical characteristics of severe respiratory syncytial virus (RSV) in neonatal period and early infancy and provide information in clinical practice. Methods : Twelve neonates and young infants (<6 months) who were infected by respiratory syncytial virus and required mechanical ventilation between March 2005 and July 2007 were enrolled. Diagnosis of RSV infection was made based on the positive results by rapid antigen immunoassay or polymerase chain reaction. Results : There were four premature infants, of whom three were near-term. Birth weight of subject patients was $2.8{\pm}0.6kg$, gestational age was $37{\pm}2weeks$ and the age at the time of admission was $35{\pm}15days$. Nine of them showed apnea and in five patients, apnea itself was an indication for mechanical ventilation. In seven of the apneic patients, apnea was the first manifestation of RSV infection. In three of these seven apneic patients, apnea preceded definite respiratory distress signs or typical stethoscopic findings by 1-3 days. Mean duration of mechanical ventilation was $3{\pm}2days$, and mean duration of stay in intensive care unit was $6{\pm}2days$. Conclusion : RSV is a major cause of severe respiratory tract infection in term or near-term infant younger than 2 months. For apnea could be the first manifestation of the RSV infection, high level of suspicion is required in practice of neonates or young infants who show any upper respiratory infection symptoms during RSV season.

Loss of Heterozygosity on the Long Arm of Chromosome 21 in Non-Small Cell Lung Cancer (비소세포폐암에서 21q 이형체 소실)

  • Chai, Po-Hee;Bae, Nack-Cheon;Lee, Eung-Bae;Park, Jae-Yong;Kang, Kyung-Hee;Kim, Kyung-Rok;Bae, Moon-Seob;Cha, Seung-Ik;Chae, Sang-Chul;Kim, Chang-Ho;Jung, Tae-Hoon
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.6
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    • pp.668-675
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    • 2001
  • Background : Non-smalll lung cancer(NSCLC) develops as a result of the accumulation of multiple genetic abnormalities. Loss of heterozygosity(LOH) is one of the most frequent genetic alterations that is found in NSCLC, and the chromosomal regions that display a high rate of LOH are thought to harbor tumor suppressor genes(TSGs). This study was done to determine the frequency of LOH in 21q with the aim of identifying potential TSG loci. Method : Thirty-nine surgically resected NSCLCs were analysed. Patients peripheral lymphocytes were used as the source of the normal DNA. Five microsatellite Inarkers of 21q were used to study LOH : 21q21.1(D21S1432, and D21S1994); 21q21.2-21.3(D21S1442) ; 21q22.1(21S1445) ; and 21q22.2-22.3(D21S266). The fractional allelic loss(FAL) in a tumor was calculated as the ratio of the number of markers showing LOH to the number of informative markers. Result : LOH for at least one locus was detected in 21 of 39 tumors(53.8%). Among the 21 tumors with LOH, 5(21.8%) showed LOH at almost all informative loci. Although statistically not significant, LOH was found more frequently in squamous cell carcinomas(15 of 23, 65.2%) than in adenocarcinomas(6 of 16, 37.5%). In the squamous cell carcinomas the frequency of LOH was higher in stage II-III (80.0%) than in stage I (53.8%). The FAL value in squamous cell carcinomas($0.431{\pm}0.375$) was significantly higher than that found in adenocarcinomas($0.l92{\pm}0.276$). Conclusion : These results suggest that LOH on 21q may be involved in the development of NSCLC, and that TSG(s) that contribute to the pathogenesis of NSCLC may exist on 21q.

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Genetic Variation of some Patches of Eleutherococcus senticosus (Rupr. & Maxim) Maxim. in Korea (국내(國內) 가시오갈피 군락(群落)의 유전변이(遺傳變異) 분석(分析))

  • Hong, Kyung-Nak;Cho, Kyung-Jin;Park, Yew-Heon;Hur, Sung-Du;Hong, Yong-Pyo;Kang, Bum-Yong
    • Journal of Korean Society of Forest Science
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    • v.89 no.5
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    • pp.645-654
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    • 2000
  • The aim of this study was to described the genetic structure of Eleutherococcus senticosus in Korea. We investigated 10 patches, which are eight Korean patches and two foreign patches come from Russia and China growing at Korean habitat, using ISSR(inter-simple sequence repeats) markers. In ISSR PCR, the overall percentage of polymorphic ISSR amplicons was 76% and the mean number of amplicons per ISSR primer was 11.5, which were higher than the RAPD results for the some cultivars collected in Korea(Kim et al., 1998) ; 57% and 5.7, respectively. So ISSR markers provide more powerful tool than RAPD markers for the investigation of genetic variation in E. senticosus. There are relatively high genetic variation among patches as 62.8%, but low variation within eight Korean patches. Such pattern of genetic variation, which is not ordinary in other tree species, may be result from the narrow and limited habitats and the asexual reproduction of this species at the natural stands in Korea. Although the small sample size in this study seemed to be resulted in the high genetic variation among patches, the overall genetic interpretation of this study might not be much affected on the basis of the characteristics of the distribution and the reproduction system of E. senticosus. Analysis of genetic distance between all pairs of the patches did not reveal any trends with regard to geographic distance, which was confirmed by the results obtained from AMOVA(analysis of molecular variance) and PCA(principal component analysis). These results suggest that, in addition to the preservation of the natural stands, the conservation of larger number of patches with small number of individuals per patch is more effective for the ex situ conservation and for maintaining the genetic diversity of E. senticosus in Korea than smaller patches with large number of individuals.

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Hypoxia Inducible Factor-1α Directly Regulates Nuclear Clusterin Transcription by Interacting with Hypoxia Response Elements in the Clusterin Promoter

  • Park, Jeongsook;Park, So Yun;Shin, Eunkyung;Lee, Sun Hee;Kim, Yoon Sook;Lee, Dong Hoon;Roh, Gu Seob;Kim, Hyun Joon;Kang, Sang Soo;Cho, Gyeong Jae;Jeong, Bo-Young;Kim, Hwajin;Choi, Wan Sung
    • Molecules and Cells
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    • v.37 no.2
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    • pp.178-186
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    • 2014
  • Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) bound directly to these sites and activated transcription. Exposure to the hypoxia-mimetic compound $CoCl_2$, incubation under 1% $O_2$ conditions, or overexpression of HIF-$1{\alpha}$ enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with $CoCl_2$ and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-$1{\alpha}$ to HRE sites and is epigenetically controlled by methylation of its promoter region.

Research status of the development of genetically modified papaya (Carica papaya L.) and its biosafety assessment (GM 파파야 개발 및 생물안전성 평가 연구 동향)

  • Kim, Ho Bang;Lee, Yi;Kim, Chang-Gi
    • Journal of Plant Biotechnology
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    • v.45 no.3
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    • pp.171-182
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    • 2018
  • Papaya (Carica papaya L.) is one of the crops widely planted in tropical and subtropical areas. The papaya fruit has low calories and are plentiful in vitamins A and C and in minerals. A major problem in papaya production is a plant disease caused by the papaya ringspot virus (PRSV). The first PRSV-resistant GM papaya expressing a PRSV coat protein gene was developed by USA scientists in 1992. The first commercial GM papaya cultivars derived from the event was approved by the US government in 1997. Development of transgenic papayas has been focused on vaccine production and limited agricultural traits, including insect and pathogen resistance, long shelf life, and aluminum and herbicide tolerance. Approximately 17 countries, including the USA and China, produced transgenic papayas and/or commercialized them, which provoked studies on biosafety assessment and development of GM-detection technologies. For the biosafety assessment of potential effects on human health, effects of long-term feeding to model animals have been studied in terms of toxicity and allergenicity. Studies on environmental safety assessment include influence on soil-microbial biodiversity and transfer to soil bacteria of GM selection markers. Many countries, such as Korea, the European Union, and Japan, that have strict regulations for GM crops have serious concerns about unintended introduction of GM cultivars and food commodities using unauthorized GM crops. Transgene- and/or GM event-specific molecular markers and technologies for genomics-based detection of unauthorized GM papaya have been developed and have resulted in the robust detection of GM papayas.

Characters of motile aeromonads isolated from imported ornamental fish (수입 관상어에서 분리한 motile aeromonads의 특성)

  • Jin, Se-yun;Ko, Chang-yong;Lee, Ye-ji;Jung, Yun-hee;Ju, Seong-cheol;Kim, Eunheui
    • Journal of fish pathology
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    • v.30 no.2
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    • pp.89-96
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    • 2017
  • The majority of freshwater ornamental fish are imported and distributed domestically, causing high risk of exposure to exotic pathogens and drug resistant bacteria in Korea. Aeromonas hydrophila is known as a common species of fresh water bacteria and opportunistic fish pathogen, as well as a species causing zoonotic infection. In this study, we isolated motile aeromonads from various imported freshwater ornamental fish and studied the characters of the isolates. Imported freshwater ornamental fish were purchased on day 1 after the fish were deposited in the aquarium. Bacteria were isolated from the liver, kidney and spleen of fish using 0.5% NaCl containing tryptic soy agar medium. Bacteria were grouped on the basis of their morphological characteristics. The colonies with clear zone on starch-ampicillin agar (SA agar) were tentatively identified as Aeromonas spp. Two hundred and twenty-six strains, about 70% of total isolates were assumed to be Aeromonas spp. Nine isolates were further identified based on the result of the API 20E test and PCR using primers specific for A. hydrophila 16S rRNA gene. The isolates were identified as A. hydrophila and the API 20E test showed differences in trisodium citrate, D-sucrose, D-melibiose, amygdalin and L-arabinose availability between the nine isolates and standard A. hydrophila. The susceptibilities of the isolated bacteria to 10 antibacterial agents were confirmed by the disk diffusion method. Isolated strains were found to be resistant to amoxicillin and ampicillin and sensitive to florfenicol. However, 7 isolates showed multiple drug resistances to erythromycin, oxytetracycline, nalidixic acid etc. Pathogenicity of the isolates was determined by the artificial challenge test on goldfish (Carassius auratus). Three isolates caused 60 ~ 80% mortality in goldfish within 5 days after the initiation of challenge. These results indicate that multiple drug resistant, highly pathogenic and exotic A. hydrophila can spread to domestic aquarium and the preventive treatment of fish before sale is necessary.