• Molecules and Cells
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• 제45권3호
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• pp.158-167
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• 2022

Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.

• 한국어병학회지
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• 제6권2호
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• pp.103-110
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• 1993

Sau3AI으로 부분절단한 Serratia marcescens genomic DNA(5Kb 이상)을 pUC19의 BamHI site에 삽입하여 total genomic library를 준비하였다. Swollen colloidal chitin media에서 halo를 형성하는 2개의 E.coli 형질전환주를 선별하였다. 이들 colony가 chitinase 유전자를 갖음을 재확인하기 위하여 4-methylumbelliferyl N-acetyl-$\beta$-D-glucosaminide(4-MuFGlcNAc)를 이용하였다. 4-MuFGlcNAc는 chitinase에 대한 기질특이성을 나타내며 형광을 나타내는 기질로서 positive clone들은 360nm의 자외선을 조사하였을 경우 밝은 형광을 나타낸다. pUC19으로 부터 유래된 2 종류의 다른 chitinase clone, pCH1(11.0Kb) 및 pCH2(7.5Kb)를 genomic DNA library로 부터 분리하였으며, 이들의 제한효소지도를 작성한 결과 서로 다른 제한효소지도를 나타내었다. pCH1EA 및 pCH2로 부터 각각의 EcoRI-Xbal fragment를 subcloning함으로써 두개의 다른 chitinase 유전자의 위치를 결정하였다. pCH1EA 및 pCH2를 cross hybridization 한 결과 hybridization signal을 나타내지 않아 서로 유사성이 없는 것으로 사료된다.

• Molecules and Cells
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• 제44권9호
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• pp.627-636
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• 2021

The three-dimensional organization of chromatin and its time-dependent changes greatly affect virtually every cellular function, especially DNA replication, genome maintenance, transcription regulation, and cell differentiation. Sequencing-based techniques such as ChIP-seq, ATAC-seq, and Hi-C provide abundant information on how genomic elements are coupled with regulatory proteins and functionally organized into hierarchical domains through their interactions. However, visualizing the time-dependent changes of such organization in individual cells remains challenging. Recent developments of CRISPR systems for site-specific fluorescent labeling of genomic loci have provided promising strategies for visualizing chromatin dynamics in live cells. However, there are several limiting factors, including background signals, off-target binding of CRISPR, and rapid photobleaching of the fluorophores, requiring a large number of target-bound CRISPR complexes to reliably distinguish the target-specific foci from the background. Various modifications have been engineered into the CRISPR system to enhance the signal-to-background ratio and signal longevity to detect target foci more reliably and efficiently, and to reduce the required target size. In this review, we comprehensively compare the performances of recently developed CRISPR designs for improved visualization of genomic loci in terms of the reliability of target detection, the ability to detect small repeat loci, and the allowed time of live tracking. Longer observation of genomic loci allows the detailed identification of the dynamic characteristics of chromatin. The diffusion properties of chromatin found in recent studies are reviewed, which provide suggestions for the underlying biological processes.

• 한국자기공명학회논문지
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• 제2권2호
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• pp.141-151
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• 1998

Human annexin I is a member of annexin family of calcium dependent phospholipid binding proteins, which have been implicated in various physiological roles including phospholipase A2(PLA2) inhibition, membrane fusion and calcium channel activity. In this work, the structure of N-terminally truncated human annexin I ({{{{ DELTA }}-annexin I) and its interactions with Ca2+, ATP and cAMP were studied at atomic level by using nuclear magnetic resonance (NMR) spectroscopy. The effect of Ca2+ binding on the structure of {{{{ DELTA }}-annexin I was investigated. The addition of Ca2+ to {{{{ DELTA }}-annexin I caused some changes in 13C NMR spectra. Carbonyl carbon resonances of some histidines were significantly broadened by Ca2+ binding. However, in the case of methionine, phenylalanine, and tyrosin, small changes could be observed. We found that ATP and cAMP bind {{{{ DELTA }}-annexin I, and the binding ratio of ATP to {{{{ DELTA }}-annexin I is 1. These results are well consistent with the report that cAMP and ATP interact with annexin I, and affect the calcium channels formed by annexin I. Because {{{{ DELTA }}-annexin I is a large protein with 35 kDa molecular weight, site-specific (carbonyl-13C) labeling technique was used to study the interaction sites of {{{{ DELTA }}-annexin I with Ca2+. NMR study was focused on the carbonyl carbon resonances of tyrosine, phenylalanine, methionine and histidine residues of {{{{ DELTA }}-annexin I because the number of these amino acids is small in the amino acid sequence of {{{{ DELTA }}-annexin I.

• Journal of Ginseng Research
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• 제34권1호
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• pp.41-46
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• 2010

Expensive herbs such as ginseng are always a possible target for fraudulent labeling. New mountain ginseng strains have occasionally been found deep within mountain areas and commercially traded at exorbitant prices. However, until now, no scientific basis has existed to distinguish such ginseng from commonly cultivated ginseng species other than by virtue of being found within deep mountain areas. Polymerase chain reaction (PCR) analysis of the internal transcribed spacer has been shown to be an appropriate method for the identification of the most popular species (Panax ginseng) in the Panax ginseng genus. A single nucleotide polymorphism (SNP) has been identified between three newly found mountain ginseng (KGD4, KGD5, and KW1) and already established Panax species. Specific PCR primers were designed from this SNP site within the sequence data and used to detect the mountain ginseng strains via multiplex PCR. The established multiplex-PCR method for the simultaneous detection of newly found mountain ginseng strains, Korean ginseng, and foreign ginseng in a single reaction was determined to be effective. This study is the first report of scientific discrimination of "mountain ginsengs" and describes an effective method of identification for fraud prevention and for uncovering the possible presence of other, cheaper ginseng species on the market.

• 대한방사성의약품학회지
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• 제5권1호
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• pp.11-17
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• 2019

Recently, antibody-like scaffold proteins have received a great deal of interest in diagnosis and therapy applications because of their intrinsic features that are often required for tumor imaging and therapy. Intrinsic issues that are associated with therapeutic application of antibody-like scaffold proteins, particularly in cancer treatment, include an efficient and straightforward radiolabeling for understanding in vivo biodistribution and excretion route, and monitoring therapeutic responses. Herein, we report an efficient and straightforward method for radiolabeling of antibody-like scaffold proteins with the $[^{99m}Tc(OH_2)_3(CO)_3]^+$ ($^{99m}Tc$-tricarbonyl) by using a site-specific direct labeling method via hexahistidine-tag, which is a widely used for general purification of recombinant proteins with His-affinity chromatography. Repebody is a new class of antibody-like scaffold protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine ($His_6$)-tag bearing repebody (rEgH9) was labeled with [$^{99m}Tc$]-tricarbonyl. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound was more than 99% pure after purification. These results clearly demonstrate that the present radiolabeling method will be useful molecular imaging study.

• 한국식품위생안전성학회지
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• 제25권2호
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• pp.162-169
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• 2010

식품위생법개정에 의한 음식점 식육 원산지 표시제가 시행됨에 따라 표시제도의 정착을 위해 마련한 과학적 한우판별 시험법을 이용하여 소고기 원산지 표시제에 대한 실패를 전국적 규모로 점검하였다. 본 연구에 사용한 한우판별시험법은 앞선 사업에서 검증된 90개의 한우판별용 SNP 바이오마커를 이용한 시험방법으로 소고기원산지표시제의 제도정착에 큰 기여를 하고 있다. 2009년도 식품안전관리지침에 계획도니 음식점 소고기 원산지 표시제 시행 대상 음식점으로는 구이용 쇠고기 판매 음식점으로서 영업장 면접이 $100m^2$이상인 곳 가운데 서울지역 48개 시료 및 지방 168개 시료 등 총 216건에 대하여 원산지 표시실태 모니터링 검사를 실시하였으며, 그 결과 총 검체의 1.3% (3건/216건)가 허위표시임이 파악되었다. 이는 2008년도의 모니터링 검사를 통하여 확인한 허위표시 비율인 5.14%에 비해 감소한 결과로 "음식점식육원산지표시제"가 점차 정착되고 있는 것으로 판단된다. 또한, 한우판별시험법이 마련되기 전 실시했던 음식점 소고기 원산지 표시에 대한 모니터링 실시결과 2005년 34.0%, 2006년 30.1%으로 나타난 반면 한우판별시험법이 확립된 후 모니터링 결과는 2007년도 3.2%, 2008년도 5.14%으로 나타나 한우판별시헙법의 확립이 음식점 소고기 원산지 표시제에 큰 기여를 하고 있음을 증명해 주고 있다.

• Radiation Oncology Journal
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• 제17권1호
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• pp.1-8
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• 1999

목적 : 실험적으로 p53 종양억제유전자는 세포의 방사선에 대한 반응을 조절하는 것으로 알려져 있는데, 임상에서 p53의 변화와 방사선치료 후의 예후와의 상호관련성은 아직 명확하게 규명되지 않은 상태이다. 이에 두경부종양환자에서 흔히 관찰되는 p53의 변화가 방사선치료결과에 어떤 영향을 미칠 수 있는지를 알아보고자 하였다. 재료 및 방법 : 두경부종양으로 진단되어 근치적 방사선치료를 받은 55명의 환자를 대상으로 임상결과를 후향적으로 분석하였다. 각 환자의 치료전 종양조직의 paraffin section을 human p53단백질에 대한 monoclonal antibody (D-07)로 면역조직화학염색하여 labeling Index (number of labelded nuclei/total number of counted nuclei x100)를 구하여, 임상결과와 연관지어 분석하였다. 결과 : 전체환자의 67.2$\%$에서 p53의 기능이상을 시사하는 과발현 소견을 보였다. 원발병소에 따른 과발현 빈도는 oral cavity, larynx, hypopharynx, nasopharynx순으로 각각 100$\%$, 76$\%$, 67$\%$, 67$\%$, 38$\%$로 나타났다. 흡연자가 비흡연자에 비해 유의하게 높은 과발현 빈도를 보였다 (78.6$\%$, 30.8$\%$). 원발병소, 병기 및 Karnofsky peformance status가 방사선치료에 대한 반응율과 유의한 연관을 보였으며, p53의 과발현여부는 치료반응율에 유의한 영향을 미치지 못하는 것으로 나타났다. 무병생존율 및 전체생존율에 영향을 미치는 인자는 원발병소와 병기였고, p53의 과발현여부는 유의한 연관을 보이지 못하였다. 결론 : 근치적 방사선치료를 받은 두경부종양 환자에서, 면역조직화학염색에 의한 p53의 과발현율은 원발병소, 병기 및 흡연여부와 유관하였으며, 과발현여부가 치료반응율 및 생존율에 유의한 영향을 미치지 못하였다.