• 대한임상검사과학회지
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• 제39권2호
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• pp.113-121
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• 2007

선천성난청은 비교적 흔한(1/1,000) 유전성질환으로 여러 유전자와 관련이 있으며, 최근에는 connexin 26 단백질내의 GJB2 유전자 돌연변이와의 관련성이 보고되고 있다. 유전질환을 예측하기 위한 유전자선별검사를 임상에 적용하기 위해서는 각 해당 국가별로 정상인에서 유전자돌연변이의 빈도를 구하고, 환자의 결과를 비교하여 활용성을 검토한 후 사용하여야 한다. 본 연구에서는 청력검사(TEOAE)가 정상인 신생아에서 GJB2 유전자 돌연변이 빈도를 구하여 screening test를 위한 한국인의 database를 수립하고자 하였다. 검체로는 437 명의 건강한 신생아의 발꿈치를 천자하여 얻은 혈액을 이용하였고, DNA는 Intron 사의 킷트를 사용하여 추출하였으며, GJB2 PCR을 실시한 후 증폭산물(783 bps)은 2% agarose gel로 전기영동을 실시하였고, DNA 자동염기서열분석기를 이용하여 분석을 실시하였다. 총 437명의 한국인 신생아에서 GJB2 유전자 중 8곳의 돌연변이(35delG, 167delT, 235delC, V27I, V37I, M34T, E114G, I203T)를 분석하였으며, 이 중 5곳에서 돌연변이가 발견되었다. 총 437명 중 301명(68.9%)에서 GJB2 유전자돌연변이가 발견되었는데, 그 중 154명이 단일돌연변이였다. V27I 변이가 271명으로 가장 많이 발견되었으며, 대부분의 V27I 변이는 E114G 변이와 함께 나타났다. E114G 변이는 총 146명, I203T 변이는 17명, V37I 변이는 14명, 235delC 변이는 1명의 순으로 나타났다. 이중돌연변이의 대부분은 V27I/E114G였으며, V27I/I203T는 3명에서 나타났고, 삼중돌연변이 V27I/E114G/I203T는 1명에서 발견되었다. 본 연구결과, PCR을 이용한 자동염기분석검사는 GJB2 유전자의 돌연변이 검출에 매우 유용하며, 본 결과는 향 후 신생아 난청선별검사를 위한 한국인의 database로 활용될 수 있을 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.31-38
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• 2016

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked $IFN-{\gamma}$. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, $5{\mu}M$) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine ($5{\mu}M$) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, $20{\mu}M$) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, $10{\mu}M$) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.

• Journal of Korean Neurosurgical Society
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• 제49권1호
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• pp.13-19
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• 2011

Objective: The purpose of this study is to investigate the combined effects of ginkgo biloba extract, ginkgolide A and B and aspirin on SK-N-MC, human neuroblastoma cell viability and mRNA expression of growth associated protein43 (GAP43), Microtubule-associated protein 2 (MAP2), B-cell lymphoma2 (Bcl2) and protein53 (p53) gene in hypoxia and reperfusion condition. Methods: SK-N-MC cells were cultured with Dulbecco's Modified Eagle's Medium (DMEM) media in $37^{\circ}C$, 5% $CO_2$ incubator. The cells were cultured for 8 hours in non-glucose media and hypoxic condition and for 12 hours in normal media and $O_2$ concentration. Cell survival rate was measured with Cell Counting Kit-8 (CCK-8) reagent assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to estimate mRNA levels of GAP43, MAP2, Bcl2, and p53 genes. Results: The ginkgolide A and B increased viable cell number decreased in hypoxic and reperfused condition. The co-treatment of ginkgolide B with aspirin also increased the number of viable cells, however, there was no additive effect. Although there was no increase of mRNA expression of GAP43, MAP2, and Bcl2 in SK-N-MC cells with individual treatment of ginkgolide A, B or aspirin in hypoxic and reperfused condition, the co-treatment of ginkgolide A or B with aspirin significantly increased GAP43 and Bcl2 mRNA levels. In MAP2, only the co-treatment of ginkgolide A and aspirin showed increasing effect. The mRNA expression of p53 had no change in all treating conditions. Conclusion: This study suggests that the combined treatments of Ginkgo biloba extracts and aspirin increase the regeneration of neuroblastoma cells injured by hypoxia and reperfusion.

• Food Science and Biotechnology
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• 제18권5호
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• pp.1100-1108
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• 2009

Lactic acid bacteria (LAB) isolated from various fruits and vegetables were screened in order to determine appropriate fermentation starters for manufacturing functional fermented onion juice. From the initial screening test comprising more than 700 isolated LAB, 16 isolates were selected based on their acid production rate. Among the selected isolates, the fermentation broth of KC-007 exhibited the highest electron donating and nitrite scavenging activities, with values at pH 1.2 of 95.6 and 68.7%, respectively. From the overall results obtained in this study, we finally selected the bacterium KC-007 as a fermentation starter. This bacterium was identified and named as Pediococcus pentosaceus based on its morphological and physiological characteristics, carbon-utilization pattern (as assessed using an API 50CHL kit), and molecular genetic characteristics (as assessed using the nucleotide sequence of the 16S rRNA gene). The optimal temperature, pH, and starter inoculation concentration (v/v) required for growth of the isolated strain were $40^{\circ}C$, pH 4.0-6.0, and 2%(v/v), respectively.

• 대한의생명과학회지
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• 제22권3호
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• pp.83-97
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• 2016

The measurement of viral load in HIV-1 infected patients is essential for the establishment of a therapeutic strategy. Several commercial assays have shown shortcomings in quantifying rare genotypes of HIV-1 such as minor groups of N and O. In this study, the HIV-1 RT-qPCR assay was developed. The primers and probe of HIV-1 were designed to target the pol gene and to increase the detection efficiency of various subtypes including group N and O. The HIV-1 quantitative RT-qPCR assay was assessed for its analytical performance and clinical evaluation. The LoD was determined to 33.9 IU/ml. The LoD of several subtypes including A, C, D, CRF_01AE, F, CRF_02AG, G and H, were determined to less than 40 IU/ml. The HIV-1 quantitative RT-qPCR assay was evaluated using the China National Reference Panel of HIV-1 RNA to determine the analytical performance. The results were all within the acceptable range. The clinical evaluation was performed at Hunan CDC in China. The clinical evaluation results were compared with those of the China domestic commercial kit. A significant correlation (fresh samples; $R^2=0.84$, P<0.001, frozen samples; $R^2=0.76$, P<0.001) between the two systems was observed for 64 fresh samples and 76 frozen samples with viral loads, and the Bland-Altman plot showed good agreement (98.4%, 96.1%, respectively). In conclusion, the HIV-1 quantitative RT-qPCR assay had comparable analytical performance with several commercial kits. The study provides basic data for the research of HIV-1 diagnosis and the development of P < HIV-1 molecular diagnostic assay.

• 한국축산식품학회지
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• 제27권4호
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• pp.509-516
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• 2007

Starters of lactic acid bacteria(LAB) were isolated from the commercial yoghurt products and the four isolates have been studied on their identification and some physiological characteristics. For the purpose of identification, microscopic examination, API test, and 16s rRNA gene sequencing were conducted. Isolate A from a yogurt product of local dairy company A was shown to be Gram-positive rod-shaped bacterium. All strains isolated were turned out to be as Lactobacillus paracasei by using a API 50 CHL kit. In contrast, isolate A was identified as a strain of Lactobacillus helveticus based on the 16S rRNA sequencing data, and L. casei ssp. casei for both B and D and L. paracasei for C. All the isolates survived the simulated gastric juice, pH 2.0 within 3 hours and sharply decreased in viability so that no viable cell was observed after 4.5 hours incubation. In addition, the four isolated strains were almost identical in antibiotic susceptibility to six different kinds of antibiotics including erythromycin ($15\;{\mu}g$), ampicillin ($10\;{\mu}g$), gentamycin ($10\;{\mu}g$), neomycin ($30\;{\mu}g$), but rather resistant to colistin ($10\;{\mu}g$) and streptomycin ($10\;{\mu}g$). It was noteworthy that four isolates were confirmed to produce antibacterial substance against foodborne pathogens of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli 0157:H7 as test organisms based on the inhibitory zones on an MRS soft agar medium. At presence, the inhibitory factor is unknown so that further studies are required to ascertain the active factor responsible for the inhibitory activities.

• 한국축산식품학회지
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• 제33권1호
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• pp.75-82
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• 2013

This study aimed to isolate lactic acid bacteria (LAB) from Kimchi and to identify suitable probiotic strain for application in fermented dairy product as a commercial starter culture. A total of 106 (LAB) strains were isolated from Kimchi collected from different regions in Korea and their phenotypic characteristics were assayed. Four isolates from MRS agar plates were selected and designated as DKL109, DKL119, DKL121 and DKL128. They were identified first by API 50 CHL kit and then 16S rRNA gene sequencing. DKL121 and DKL128 were identified as Lactobacillus paracasei and Lactobacillus casei, respectively. Other two isolates (DKL109 and DKL119) were identified as Lactobacillus plantarum. To estimate their applicability in dairy products, the characteristics including acid and bile tolerance, cold shock induced cryotolerance and enzymatic activities were determined. There was wide variation in ability of strains to acid tolerance, but no significant differences in bile tolerance, cold shock induced cryotolerance within selected strains. DKL119 and DKL121 showed the highest resistance to acid and bile and the highest ${\beta}$-galactosidase activity, respectively. When these two strains were used for yogurt preparation as a single starter culture, their viable cell counts reached to $1.0{\times}10^9CFU/mL$. Lactobacillus plantarum DKL119 showed faster acid development than commercial starter culture. Also storage trials at $10^{\circ}C$ showed that the viability of these strains was retained over 15 d. With these results, it was indicated that probiotics isolated from Kimchi can be used in yogurt manufacturing as a starter culture.

• Clinical and Experimental Reproductive Medicine
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• 제51권1호
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• pp.28-41
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• 2024

Objective: Chronic scrotal hyperthermia (SHT) can lead to serious disorders of the male reproductive system, with oxidative stress playing a key role in the onset of these dysfunctions. Thus, we evaluated the impact of caffeine, a potent antioxidant, on cellular and tissue disorders in mice with chronic SHT. Methods: In this experimental study, 56 adult male NMRI mice were allocated into seven equal groups. Apart from the non-treated control group, all were exposed to heat stress. Two groups, termed "preventive" and "curative," were orally administered caffeine. The preventive mice began receiving caffeine immediately prior to heat exposure, while for the curative group, a caffeine regimen was initiated 15 consecutive days following cessation of heat exposure. Each treated group was subdivided based on pairing with a positive control (Pre/ curative [Cur]+PC) or a vehicle (Pre/Cur+vehicle). Upon conclusion of the study, we assessed sperm characteristics, testosterone levels, stereological parameters, apoptosis, antioxidant and oxidant levels, and molecular markers. Results: Sperm parameters, testosterone levels, stereological parameters, biochemical factors (excluding malondialdehyde [MDA]), and c-kit gene expression were significantly elevated in the preventive and curative groups, especially the former, relative to the other groups. Conversely, expression levels of the heat shock protein 72 (HSP72) and nuclear factor kappa beta (NF-κβ) genes, MDA levels, and apoptotic cell density were markedly lower in both caffeine-treated groups relative to the other groups, with more pronounced differences observed in the preventive group. Conclusion: Overall, caffeine attenuated cellular and molecular abnormalities induced by heat stress in the testis, particularly in the mice treated under the preventive condition.

• Journal of Korean Neurosurgical Society
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• 제48권1호
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• pp.20-30
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• 2010

Objective : We determined whether the expression of GRIM-19 is correlated with pathologic types and malignant grades in gliomas, and determined the function of GRIM-19 in human gliomas. Methods : Tumor tissues were isolated and frozen at $-80^{\circ}C$ just after surgery. The tissues consisted of normal brain tissue (4), astrocytomas (2), anaplastic astrocytomas (2), oligodendrogliomas (13), anaplastic oligodendrogliomas (11), and glioblastomas (16). To profile tumor-related genes, we applied RNA differential display using a $Genefishing^{TM}$ DEG kit, and validated the tumor-related genes by reverse transcription polymerase chain reaction (RT-PCR). A human glioblastoma cell line (U343MG-A) was used for the GRIM-19 functional studies. The morphologic and cytoskeletal changes were examined via light and confocal microscopy. The migratory and invasive abilities were investigated by the simple scratch technique and Matrigel assay. The antiproliferative activity was determined by thiazolyl blue Tetrazolium bromide (MTT) assay and FACS analysis. Results : Based on RT-PCR analysis, the expression of GRIM-19 was higher in astrocytic tumors than oligodendroglial tumors. The expression of GRIM-19 was higher in high-grade tumors than low-grade tumors or normal brain tissue; glioblastomas showed the highest expression. After transfection of GRIM-19 into U343MG-A, the morphology of the sense-transfection cells became larger and more spindly. The antisensetransfection cells became smaller and rounder compared with wild type U343MG-A. The MTT assay showed that the sense-transfection cells were more sensitive to the combination of interferon-$\beta$ and retinoic acid than U343MG-A cells or antisense-transfection cells; the antiproliferative activity was related to apoptosis. Conclusion : GRIM-19 may be one of the gene profiles which regulate cell death via apoptosis in human gliomas.

• 생명과학회지
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• 제21권12호
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• pp.1726-1731
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• 2011

본 연구의 목적은 누에형질전환체를 이용하여 재조합단백질 대량생산 시스템을 개발하는 것으로서, 본 실험에서는 hSCF유전자를 이용하여 누에에서 재조합단백질을 생산하였다. 실험에 사용된 piggyBac 전이벡터는 hSCF 유전자의 발현 조절을 위해 초파리 유래의 dHsp70 promoter를 사용하였고, EGFP marker유전자는 3xP3 promoter로 발현을 조절하였다. 총 1,020 개의 누에알에 microinjection 하여 G1 세대에서 22 bloods의 형질전환체를 선발하였고, 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 GFP 형광을 관찰 할 수 있었다. hSCF 재조합단백질의 발현은 Western blot 분석으로 확인 할 수 있었고, inverse PCR 분석을 통해서 누에 게놈에 전이벡터가 삽입된 것을 확인할 수 있었다. 지금까지의 실험 결과에서 hSCF 재조합 단백질이 누에에서 생산될 수 있음을 확인 할 수 있었다. 비록 누에에서 생산된 hSCF 재조합단백질의 생리활성에 대한 실험이 추후에 요구되지만, 이러한 실험결과는 piggyBac 전이벡터와 microinjection 법으로 누에에서 고부가가치의 재조합단백질을 대량생산 할 수 있음을 보여 주었다고 할 수 있겠다. 따라서 누에를 유용물질 생산을 위한 생체반응기로서 활용할 수 있을 것으로 기대된다.