• Clinical and Experimental Pediatrics
• /
• 제45권3호
• /
• pp.302-310
• /
• 2002

목 적: 본 연구에서는 임상적으로 XLA로 진단받고 현재 치료 중인 국내 환아 세가족의 네명의 환아와 모친 등을 대상으로 말초혈액 단핵구의 Btk 단백질 발현과 Btk 유전자 변이를 분석하고자 하였다. 방 법: 말초혈액 단핵구의 Btk 발현도를 항 Btk 항체를 이용한 유세포측정을 통해 분석하고 직접 염기서열 분석에 의해 Btk 유전자 변이를 분석하였다. 결 과 : 환아들의 B 림프구의 발현은 2.1% 미만으로 정상인에 비해 매우 저하되어 있었으며 유세포 측정에 의한 환아 단핵구 유래 Btk 단백질 발현도 1.0% 이하로 정상인에 비해 매우 감소되었다. 유전자 변이 분석 결과 XLA 가족 1과 3에서는 각각 intron 18과 intron 1의 짜집기 공여 위치 부위에서 1개의 점 돌연변이가 발견되었으며 cDNA상 짜집기 오류 현상을 야기하였다. 그리고 XLA 가족 2의 경우 exon 10을 포함하는 980 bp의 유전자 결손(intron 9＋191T부터 intron 10-215C까지)이 발견되었으며 세계적으로 처음 보고되는 새로운 유전자 변이였다. 또한 가족 2의 경우 가족 중 환아에서 만 Btk 단백질 결핍 및 유전자 변이를 진단하여 국내 XLA의 환아 중 최초의 산발적인 발병 양상을 확인하였다. 결 론: 본 연구 결과를 종합해 볼 때 임상적으로 XLA로 진단된 환아와 가족에 대한 항 Btk 항체를 이용한 유세포측정 방법은 XLA 환아 및 보인자의 진단에 매우 유용한 방법으로 생각된다. 또한 분자유전학 기법을 이용하여 Btk의 noncoding region을 포함한 광범위한 유전자 영역의 염기서열 분석을 시행한 결과 새로운 1개의 유전자 결손 및 2개의 점돌연변이에 의한 짜집기 오류를 확인하여 XLA를 최종 확진할 수 있었다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권7호
• /
• pp.4393-4398
• /
• 2013

Background: Chromosomal translocations are genetic aberrations associated with specific non-Hodgkin lymphoma (NHL) subtypes. This study investigated the differential gene expression profile of Egyptian NHL cases based on a microarray approach. Materials and Methods: The study included tissue samples from 40 NHL patients and 20 normal lymph nodes used as controls. Total RNA was extracted and used for cDNA microarray assays. The quantitative real time polymerase chain reaction was used to identify the aberrantly expressed genes in cancer. Results: Significant associations of 8 up-regulated and 4 down-regulated genes with NHL were observed. Aberrant expression of a new group of genes not reported previously was apparent, including down-regulated NAG14 protein, 3 beta hydroxy-delta 5-c27 steroid oxi-reductase, oxi-glutarate dehydrogenase (lipo-amide), immunoglobulin lambda like polypeptide 3, protein kinase x linked, Hmt1, and caveolin 2 Tetra protein. The up-regulated genes were Rb binding protein 5, DKFZP586J1624 protein, protein kinase inhibitor gamma, zinc finger protein 3, choline ethanolamine phospho-transferase CEPT1, protein phosphatase, and histone deacetylase-3. Conclusions: This study revealed that new differentially expressed genes that may be markers for NHL patients and individuals who are at high risk for cancer development.

• Clinical and Experimental Pediatrics
• /
• 제45권2호
• /
• pp.183-191
• /
• 2002

목 적 : 본 연구에서는 임상적으로 XLA로 진단받고 현재 치료 중인 한가족 3명 환아의 가계를 대상으로 말초혈액 단핵구의 Btk 단백질 발현과 Btk 유전자 변이를 분석하고자 하였다. 방 법 : 환아 말초혈액 단핵구의 Btk 발현도를 항 Btk 항체를 이용한 유세포계측을 통해 분석하고 PCR-SSCP 및 직접 염기서열 분석에 의해 Btk 유전자 변이를 분석하였다. 결 과 : 유세포 계측 및 PCR-SSCP에 의하여 가계내의 환자 3명 및 보인자 4명을 확인하였으며 환아들의 단핵구의 Btk 발현도를 조사한 결과 2.7% 미만으로 건강인에 비해 매우 유의한 감소를 나타내었다. 유전자 변이 분석 결과 Btk 유전자 exon 3에서 점돌연변이($T{\rightarrow}C$)가 발견되어 61번째 아미노산의 치환이 일어난 과오돌연변이를 확인할 수 있었다. 동일한 유전형을 지닌 3명의 환아들은 임상적으로 매우 다양한 표현형을 나타내었다. 특히 환아들의 혈청 IgG level이 낮을수록 임상 양상은 심한 소견을 보였다. 결 론 : 본 연구 결과를 종합해 볼 때 임상적으로 XLA로 진단된 환아와 가족에 대한 항 Btk 항체를 이용한 유세포계측 및 PCR-SSCP 방법은 XLA 환아 및 보인자의 진단에 매우 유용하며 신속하게 적용될 수 있는 기법으로 생각된다. 또한 분자유전학 기법을 이용하여 환아의 유전자 변이를 검색한 결과 Btk의 PH 도메인 내 한개의 과오돌연변이를 검색하여 XLA를 최종 확진할 수 있었다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제17권1호
• /
• pp.146-152
• /
• 2004

Ghrelin is an acylated peptide recently identified as the endogenous ligand for the growth hormone (GH) secretagogues receptor 1a (GHS-R1a) and is involved in a novel system for regulating GH release. To understand the long-term effects of ghrelin, here we constructed six myogenic expression vectors containing the cDNA of swine mature ghrelin (pGEM-wt-sGhln, pGEM-wt-hGhln), ghrelin mutant of $Ser^3$ with $Trp^3$ (pGEM-mt-sGhln, pGEM-mt-hGhln) and truncated ghrelin derivative (pGEM-tmtsGhln, pGEM-tmt-hGhln) encompassing the first 7 residues of ghrelin (including $Ser^3$ substituted with $Trp^3$) and adding a basic amino acid, Lys (K) in the C-terminus. The constructs, pGEM-wt-sGhln, pGEM-mt-sGhln and pGEM-tmt-sGhln were linked with the ghrelin leader sequence, while the pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln were linked with a leader sequence from the human growth hormone releasing hormone (hGHRH). Intramuscular injection of 200 ${\mu}g$ pGEM-wt-sGhln or pGEM-tmt-sGhln augmented growth over 3 weeks in normal rats and peaked at day 21 or 14 post-injection respectively, whose body weight gains were on average approximately 6% or 19% heavier over controls. However, other injectable vectors had no such enhanced growth effects. Our results suggested that the efficacy of the ghrelin leader sequence was more effective than that of hGHRH in our system. Moreover, the results indicated that skeletal muscle might have the ability to posttranslationally modify the in vivo expressed ghrelin. And the most strikingly, the short ghrelin analog seems to mimic the biological effects more efficiently when compared with the full-length ghrelin.

• 한국미생물·생명공학회지
• /
• 제46권3호
• /
• pp.234-243
• /
• 2018

남극해에는 산업적으로 유용한 신규 효소촉매를 생산하는 미생물들이 들어 있다. 우리는 로스해(Ross Sea)로부터 분리한 여러 저온성 박테리아를 조사하였으며, 그 중에서 지방분해 능력이 뛰어난 Croceibacter atlanticus (No. 40-F12)를 찾았다. Shotgun 클로닝 방법으로 리파아제 유전자(lipCA)를 찾았으며 Escherichia coli 균에서 LipCA 효소를 발현하였다. Spain Arreo metagenome alpha/beta hydrolase를 기준으로 LipCA 상동구조모델을 만들어서 분석한 결과, ${\alpha}/{\beta}$ hydrolase fold, Gly-X-Ser-X-Gly motif, 그리고 lid 구조를 갖고 있기 때문에 전형적인 리파아제 효소임이 밝혀졌다. Ammonium sulfate 침전법과 겔여과 크로마토그래피를 통해서 세포추출액으로부터 LipCA 효소를 순수하게 분리한 후, 최적 온도, pH, 안정성, 기질특이성, 유기용매 안정성 등의 효소특성을 규명하였다. LipCA를 cross-linked enzyme aggregate (CLEA) 방법으로 고정화하고 효소특성을 조사, 비교하였다. 고정화를 통해 온도, pH, 유기용매에 대한 안정성이 증가하였고 기질특이성의 변화는 관찰되지 않았다. $LipCA^{CLEA}$는 원심분리 방법으로 쉽게 회수되었고 4번의 재사용 후에 40% 이상의 활성이 잔재하였다. 이 논문은 C. atlanticus 리파아제의 발현, 특성규명, Cross-linked Enzyme Aggregated 고정화를 바탕으로 안정성을 높여 산업적 활용 가능성을 제시한 최초의 보고이다.

• Journal of Microbiology
• /
• 제42권2호
• /
• pp.126-132
• /
• 2004

A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30$^{\circ}C$ until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30$^{\circ}C$ for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Topl0F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, com-pared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.

• BMB Reports
• /
• 제42권4호
• /
• pp.232-237
• /
• 2009

Sterol regulatory element-binding protein (SREBP)-1c plays a crucial role in the regulation of lipogenic enzymes in the liver. We previously reported that an X-chromosome-linked RNA binding motif (RBMX) regulates the promoter activity of Srebp-1c. However, still unknown was how it regulates the gene expression. To elucidate this mechanism, we screened the cDNA library from mouse liver by yeast two-hybrid assay using RBMX as bait and identified scaffold attachment factor B1 (SAFB1). Immunoprecipitation assay demonstrated binding of SAFB1 to RBMX. Chromatin immunoprecipitation assay showed binding of both SAFB1 and RBMX to the upstream region of Srebp-1c gene. RNA interference of Safb1 reduced the basal and RBMX-induced Srebp-1c promoter activities, resulting in reduced Srebp-1c gene expression. The effect of SAFB1 overexpression on Srebp-1c promoter was found only in the presence of RBMX. These results indicate a major role for SAFB1 in the activation of Srebp-1c through its interaction with RBMX.

• 대한약침학회지
• /
• 제18권1호
• /
• pp.19-26
• /
• 2015

Objectives: Quercetin, a flavonoid compound, has been reported to induce apoptosis in cancer cells, but its anti-inflammatory effects, which are also closely linked with apoptosis, if any, on non-small-cell lung cancer (NSCLC) have not so far been critically examined. In this study, we tried to determine if quercetin had any demonstrable anti-inflammatory potential, which also could significantly contribute to inducing apoptosis in a NSCLC cell line, A549. Methods: In this context, several assays, including cytotoxicity, flow cytometry and fluorimetry, were done. Gene expression was analyzed by using a western blot analysis. Results: Results revealed that quercetin could induce apoptosis in A549 cells through mitochondrial depolarization by causing an imbalance in B-cell lymphoma 2/Bcl2 Antagonist X (Bcl2/Bax) ratio and by down-regulating the interleukine-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway. An analysis of the data revealed that quercetin could block nuclear factor kappa-light-chain-enhancer of activated B cells (NF-${\kappa}B$) activity at early hours, which might cause a down-regulation of the IL-6 titer, and the IL-6 expression, in turn, could inhibit p-STAT3 expression. Down-regulation of both the STAT3 and the NF-${\kappa}B$ expressions might, therefore, cause down-regulation of Bcl2 activity because both are major upstream effectors of Bcl2. Alteration in Bcl2 responses might result in an imbalance in the Bcl2/Bax ratio, which could ultimately bring about mitochondria mediated apoptosis in A549 cells. Conclusion: Overall, the finding of this study indicates that a quercetin induced anti-inflammatory pathway in A549 cells appeared to make a significant contribution towards induction of apoptosis in NSCLC and, thus, may have a therapeutic use such as a strong apoptosis inducer in cancer cells.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권3호
• /
• pp.275-279
• /
• 2005

Insect cell transformants, stably expressing human $GFP_{uv}-{\beta}1$, 3-N-acetylglucosaminyltransferase 2 $({\beta}3GnT2)$ as the green fluorescent protein $(GFP_{uv})-fused$ protein, were efficiently isolated on Western blot by the quantification of the densitometric intensity of the fusion protein. From almost 150 transformants containing the fusion gene linked to three different types of signal sequence, two transformants, Tn-pXme4a and -pX28a, were successfully selected, showing 8.3 and 8.6 mU/mL ${\beta}3GnT$ activity, respectively. This method requires a screening time almost one-half that required in the isolation of stably transformed cells with high expression levels, and at the same time allows the handling a large number of transformants.

• Asian Pacific Journal of Cancer Prevention
• /
• 제13권4호
• /
• pp.1471-1476
• /
• 2012

Background and Aim: Currently available systemic therapies for malignant melanoma produce low response rates in patients, and more effective treatment modalities are clearly needed. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand has a significant impact on therapy for patients with X-linked inhibitor of apoptosis protein-downregulation malignant melanoma. The primary objective of this study was to assess its therapeutic potential. Materials and Methods: We employed a conditionally replicating oncolytic adenoviral vector, named CRAd5.TRAIL/siXIAP, with the characteristics of over-expression of the therapeutic gene TRAIL and downregulation of XIAP in one vector. B16F10-luc cells were employed to detect anti-tumor activity of CRAd5.TRAIL/siXIAP in vitro and in vivo. Results: CRAd5.TRAIL/siXIAP enhanced caspase-8 activation and caspase-3 maturation in B16F10 cells in vitro. Furthermore, it more effectively infected and killed melanoma cells in vitro and in vivo than other adenoviruses. Conclusion: Taken together, the combination of upregulation of TRAIL and downregulation of siXIAP with one oncolytic adenoviral vector holds promise for development of an effective therapy for melanomas and other common cancers.