• 생명과학회지
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• 제30권11호
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• pp.1012-1020
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• 2020

생쥐의 가변부위가 인간의 정상부위에 연결되어 제조된 바이오시밀러 자연항체치료제인 레미케이드는 암괴사인자-알파(TNF-α)에 특이적인 항체로써 카이메릭 단일클론항체이며 류마티스 관절염치료를 위해 개발되었다. 바이오시밀러 레미케이드 항체의 생물학적 기능을 연구하기 위해 우리는 단백질 데이터 은행을 이용한 생물정보학 분석을 수행하여 레미케이드 자연항체와 암괴사인자-알파 항원간의 결합기작특징을 분석하였다. 자연항체를 생산하는 세포의 유전적 불안정성 때문에 레미케이드 항체생산이 제한되므로 우리는 중 사슬 가변부위를 다중펩타이드 링커에 의해 경 사슬 가변부위에 연결된 레미케이드 ScFv항체(Remicade)를 제조하였다. 더욱이 더 높은 생산과 더 높은 항원결합력을 위해 레미케이드 ScFv를 leucine zipper에 융합시켰다. Remicade와 RemicadeScZip ScFv는 대장균에서 발현되었고 Ni⁺-NTA-아가로스 컬럼으로 정제하였다. 정제된 단백질들은 예상한대로 sodium dodecyl sulfate-polyacrylamide electrophoresis에서 28.80 kDa과 33.96 kDa을 나타내었다. Remicade는 ELISA, western blot에서 TNF-α 항원에 대한 결합력이 관찰되지 않았으나 RemicadeScZip은 항원결합력을 나타내었다. 추가적인 BLI분석으로 RemicadeScZip의 TNF-α 항원에 대한 결합력을 재확인시켜주었으며 이 결과는 Leucine zipper가 레미케이드 ScFv의 접힘을 안정화시키고 TNF-α 항원에 대한 결합력을 개선시켰음을 제시해주고 있다.

• The Korean Journal of Physiology and Pharmacology
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• 제7권1호
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• pp.9-14
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• 2003

Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.

• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1376-1383
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• 2018

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity ($K_D$) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.

• 식물조직배양학회지
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• 제26권2호
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• pp.121-126
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• 1999

참나물 (Pimpinella brachycarpa)의 엽병 (petiole)절편체로부터 캘러스가 MS배지 (0.5mg/L 2,4-D와 0.1mg/L BAP)에서 유도되었으며 이들 캘러스로부터 치밀하게 배열된 세포집단(cell cluster)을 선발하여 현탁배양하였다. 이들 현탁배양세포들은 0.1 mg/L NAA가 포함된 MS고체배지에 배양되어 배발생 (embryogenic) 캘러스로 성장하였다. 배발생캘러스는 연한 노란색을 띠며 체세포배로 분화되었으며 이들 체세포배는 MS액체배지에서 발아되어 식물체로 성장하였다. 참나물 현탁배양세포 유래 배발생캘러스로부터 분리한 mRNA로부터 cDNA library를 합성하여 PCR을 수행한 결과 제조된 library의 삽입절편의 크기가 대부분 500bp이상임을 확인하였다. 이들 cDNA library로부터 전체 1.5 $\times$$10^{6}$개의 plaque를 혼성화하여 일차의 screening을 통해 19개의 cDNA clone을, 이차의 screening을 통해 5개의 cDNA clone을 얻었으며 이중 4개의 cDNA clone은 참나물 shoot의 HD-Zip 유전자인 Phz4 유전자와 동일한 약 1.4 kb 정도인 것으로 나타났으나, 1개의 cDNA clone, Phc5는 약 1.5kb정도의 크기를 나타내었다. 1.5kb인 Phc5는 Phz4유전자의 5'쪽으로 163bp의 염기가 추가로 발견되어 총 1,531 bp에 해당하였으며 18개의 polyA tail을 가지고 있었다. Phc5는 284번째에 ATG개시코돈이 있고 302개의 아미노산을 암호화하는 906개의 단백질 암호화 부위와 Homeodomain을 갖고 있었다. Phc5로부터 추정된 단백질은 기존 전사조절자에서 많이 보고된 HD의 구조적 특징을 갖고 있었다.

• Journal of Periodontal and Implant Science
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• 제31권3호
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• pp.611-623
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• 2001

Cyclosporin A is a cyclic polypeptide produced by the metabolism of fungi. It is widely used at present as immunosuppressive treatment following organ transplants. It is also used to deal with autoimmune diseases such as rheumatoid arthritis or type II diabetes. Gingival hyperplasia is one of the most frequent side-effects associated with the prescription of Cyclosporin A. The mechanisms involved in Cyclosporin A induced gingival hyperplasia are not yet clear. In vitro Cyclosporin A promotes proliferation of gingival fibroblasts, that Cyclosporin A act as a mitogen. Its action is based on mitosis of gingival fibroblasts regulated by cell cycle regulatory proteins. It was the purpose of the present study to examine the effects of Cyclosporin A on human gingival fibroblasts by means of biological and biochemical criteria. In this present study, we examined change of cell proliferation, cell activity, cell viability and cell cycle progression after application of Cyclosporin A. We also examined expression of cell cycle regulatory proteins by western blot analysis. Human gingival fibroblasts were cultured for 48 hours with application of Cyclosporin A at concentrations of 0.01, 0.1, 1, and 10 ng/ml. Cyclosporin A(1 ng/ml) significantly increased the cell activity of gingival fibroblast. Proliferation and viability of gingival fibroblasts were also increased in group treated with 1 ng/ml of Cyclosporin A compared to control group. In the cell cycle analysis, S phase was increased and G1 phase was decreased in the group treated with 1 ng/ml of Cyclosporin A. Cyclosporin A increased the expression of cdk4 and inhibited the expression of pRB and p21. These results suggest that 1 ng/ml of Cyclosporin A may increase the cell cycle progression of human gingival fibroblasts, and its mechanisms may increase the expression of cdk4 and decrease the expression of pRB and p21.

• Journal of Periodontal and Implant Science
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• 제26권2호
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• pp.429-447
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• 1996

Transforming growth factor $-{\beta}$ is one of the polypeptide growth factors that mediate the activity of mesenchymal cells and regulate wound healing process via cell proliferation, migration and extracellular matrix formation. The purposes of this study is to evaluate the effects of transforming growth factor $-{\beta}$ on the protein synthetic activity of human periodontal ligament cells and human gingival fibroblasts. The cells which were prepared were primary cultured gingival fibroblasts and periodontal ligament cells from humans, and the fourth or sixth subpassage were used in the experiments. Cells were seeded and at a confluent state, 0, 0.5, I, 2.5, 5, 10 ng/ml $TGF-{\beta}$ and $2{\mu]Ci/ml\;[^3H]$ proline were added to the cells and cultured for 24 hours. Then, 1 and 5 ng/ml concentrations were selected and added to confluent cells and cultured for 24 and 48 hours. They were labeled with $2{\mu}Ci/ml\;[^3H]$ proline for 24 hours and a collagen assay was done by the Peterkofsky and Diegelman method. The results were presented as the mean disintegration per minute (dpm) per well and S.D. of four determinations, The results were as follows. : The total protein, collagen and noncollagenous protein synthesis in periodontal ligament cells and gingival fibroblasts were increased dose- dependently by transforming growth factor-p to 2.5-5 ng/ml concentration and decreased at 10 ng/ml concentration. The percent of collagen was slightly changed according to the concentration of transforming growth factor-po The effect of transforming growth $factor-{\beta}$ was not specific for collagen synthesis since it increased the total, noncollagenous and collagenous protein, simultaneously. In the comparison of protein synthetic activity between the human periodontal ligament cells and human gingival fibroblasts, the human gingival fibroblasts had higher activities than the human periodontal ligament cells at all times and concentrations of $TGF-{\beta}$. In the comparison of protein synthetic activity between the 24 hour effect and the 48 hour effect of $TGF-{\beta}$, the 48 hour cultured cells' synthetic activity decreased more than the 24 hour cultured cells at human periodontal ligament cells and human gingival fibroblasts. In conclusion, $TGF-{\beta}$ has important roles in the stimulation of protein synthesis in human periodontal ligament cells and human gingival fibroblasts. Thus, it may be useful for clinical application in periodontal regenerative procedures.

• 한국컴퓨터정보학회논문지
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• 제16권11호
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• pp.67-76
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• 2011

본 논문은 삼차원 볼륨 영상으로부터 컨투어 트리를 이용하여 볼륨 영상의 구조를 보여주고 등위면과 그 요소들을 대화형으로 선택하고 시각화하여 영상의 깊은 이해를 돕는 새로운 기법과 프로그램의 개발에 관하여 기술한다. 이 기법의 특징은 컨투어 트리를 사용자가 이해하기 쉽게 2차원 평면상에 배치하는 새로운 알고리즘과, GPU의 병렬구조를 활용하여 등위면 요소를 효율적으로 추출할 수 있도록 하는 알고리즘을 제시한 데 있다. 이러한 알고리즘의 구현을 통해 본 연구진이 개발한 프로그램은 컨투어 트리를 이용하여 등위면 요소를 추출하는 대화형 사용자 인터페이스와 기존의 등위면 및 볼륨 렌더링이 융합된 시각화를 가능하게 해주는 특징이 있다. 본 논문에서 제안하는 기법의 우수성을 검증하기 위하여, 제안된 알고리즘을 삼차원 생의학 영상에 적용하여 그 성능을 측정해 보았다. 그 결과 제안된 컨투어 트리 배치 기법에 기반한 사용자 인터페이스를 이용하여 주어진 영상의 관심 영역인 폴리펩타이드 체인과 뇌실 그리고 대퇴골을 나타내는 등위면 요소를 각각 대화형으로 선택하고 이를 기존 방법에 비교하여 3배~4배이상 빠른 속도로 계산할 수 있었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제28권2호
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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.

• Journal of Periodontal and Implant Science
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• 제30권1호
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• pp.39-52
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• 2000

Polypeptide growth factor belong to a class of potent biologic mediator which regulate cell differentiation, proliferation, migration and metabolism. 1,25-dihydroxyvitamin $D_3$ decrease cell proliferation, and stimulate alkaline phosphatase activity which express in osteoblast during cell differentiation period. IGF-I is known to stimulate cell proliferation and differentiation too. 1,25-dihydroxyvitamin $D_3$ is known to increase IGF-I binding sites and IGF binding protein which inhibite the effect of IGF. The purpose of this study is to evaluate potential role of IGF-I as mediator that control the action of 1,25-dihydroxyvitamin $D_3$. MC3T3-E1 cell were seeded $5{\times}10^5/ml$ at 100mm culture plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 5% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ added. Total mRNA was extracted at 0, 6, 24, 48, 72 hour. PRPCR method was programed for the detection of IGF-I mRNA. In the both groups of 1,25-dihydroxy vitamin $D_3$ treated and control, alternative splicing form of IGF-I, IGF-IA and IGF-IB were expressed. In the 1,25-dihydroxyvitamin $D_3$ treated group, IGF-I mRNA expression was matained until 24 hour, there after expression was decresed. MC3T3-E1 cell were seeded $2.5{\times}10^4/ml$ at 24well plate in ${\alpha}-MEM$ containing 10% fetal bovine serum. After 48 hour incubation period, medium were changed ${\alpha}-MEM$ containing 3% fetal bovine serum. After 24 hours, $10^{-9}M$ 1,25-dihydroxyvitamin $D_3$ and 10 ng/ml IGF-I were added separately or together. Cell were cultured for 1 and 3 days, $2{\mu}Ci/ml\;[^3H]$ -thymidine was added for the last 24h of culture of each days. ${[^3H]}$-thymidine incorporation in to DNA was measured and expressed counter per minute(CPM). DNA synthetic activity was significantly decreased by 1,25-dihydroxyvitamin $D_3$ both at 1 day and 3 day, and in the combination group of 1,25-dihydroxyvitamin $D_3$ and IGF-I, DNA synthetic activity was also decreased both at 1 day and 3 days. IGF-I did not affect the DNA synthetic activity compared to control group both at 1 day and 3 day. From the above results, 1,25-dihydroxyvitamin $D_3$ was potent inhibitor of cell proliferaton in MC3T3-E1 cells. It assumed that the effect of 1,25-dihydroxyvitamin $D_3$ on osteoblast proliferation may be mediated in part by decreased level of IGF-I.

• 대한화학회지
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• 제37권4호
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• pp.378-389
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• 1993

합성 polypeptide인 poly(γ-benzyl-L-glutamate)[PBLG]를 A-segment로 하고, polyoxypropylene(POP)를 B-segment로 하는 AB 형태 블록공중합체와 polypropyleneglycol을 B-segment로 하는 ABA 형태 블록공중합체를 합성한 후, 이들 공중합체의 생식 호르몬인 progesterone, estrone, estradiol, testosterone의 고분자 매트릭스를 제조하여 이들 스테로이드의 방출량의 변화를 연구하였다. PBLG-POP1와 PBLG-POP2의 매트릭스에서의 progesterone, estradiol 및 testosterone 방출속도와 ABA 형태의 GPG1 공중합체에서의 progesterone, estrone 및 testosterone 방출속도가 염기성용액보다 산성용액에서 더 빠름을 보았다. 한편 PBLG-POP1와 PBLG-POP2의 매트릭스에서의 estrone 그리고 ABA 형태의 GPG1의 매트릭스에서의 estradiol의 방출속도는 산성용액에서 보다 중성 및 염기성용액에서 더 빨랐다. 서로 다른 매트릭스에서의 방출차이는 GPG1 > PBLG-POP1 > PBLG-POP2이며, 방출속도는 스테로이드의 분자량에 반비례함을 보였다.