• 한국미생물·생명공학회지
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• 제33권4호
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• pp.255-259
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• 2005

본 실험에서는 HRF의 조절단백질을 알아보기 위해 HRF를 bait로 한 yeast two hybrid assay를 실행한 결과 해당과정에 관여하는 TPI(triosephosphate isomerase)라는 효소를 발견하였으며, 가장 많이 중복되어 있었다. In vitro에서 HRF는 TPI의 C말단 잔기 부근(아미노산 156-249)이 상호작용에 주로 관여하는 부위임을 알 수 있었다. 또한, HeLa 세포에서 immunoprecipitation을 이용하여 HRF와 TPI의 상호작용이 실제 in vivo에서도 일어나는 현상이라는 것을 밝혔다. 결과적으로 HRF와 TPI 상호작용은 세포내 일정량이 존재하며 여러가지 신호전달에 의해 동시에 Na,K-ATPase와도 상호작용하는 것으로 생각된다.

• 미생물학회지
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• 제51권4호
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• pp.435-439
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• 2015

THO/TREX 복합체는 전사 신장, mRNA 가공 및 방출, 그리고 유전체 안정성에 중요한 역할을 담당한다. 분열효모 Schizosaccharomyces pombe에서 THO/TREX 복합체의 한 구성요소인 THOC5의 이종상동체를 암호화하고 있는 SPBC 577.04 유전자를 찾아, 그것의 기능을 분석하였다. S. pombe thoc5 (spthoc5) 유전자는 생장과 mRNA의 방출에 필수적이지는 않지만, 결실돌연변이는 야생형에 비해 생장 결함을 보였고 $poly(A)^+$ RNA도 핵 안에 약간 축적되는 현상을 보였다. 또한 정상적인 기능을 가진 spThoc5-GFP 단백질은 주로 핵안에 존재하였다. Co-immunoprecipitation 분석에서 진화적으로 잘 보존된THO/TREX 복합체의 주요 구성인자인 Hpr1(THOC1)는 또 다른 구성인자인 Tho2 (THOC2) 뿐만 아니라 spThoc5와도 상호작용을 하였다. 이와 같은 결과들은 S. pombe의 Thoc5 상동체도 THO/TREX 복합체의 구성인자로 mRNA 방출에 관여하고 있음을 시사한다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제41권
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• pp.46.1-46.9
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• 2019

Background: Oral squamous cell carcinoma (OSCC) constitutes a group of tumors that exhibit heterogeneous biology, histopathology, and clinical behaviors. Case presentation: A 73-year-old male had a whitish leukoplakia-like lesion around inflamed peri-implant area (#42, #43, and #44), and this lesion had transformed to OSCC within 3 years. He underwent mass resection, selective neck dissection, and reconstructive surgery. To detect any carcinogenesis progression, we examined the removed tumor tissue as well as the patient's preoperative and postoperative sera to identify causative oncogenic proteins using immunoprecipitation high-performance liquid chromatography (IP-HPLC). Conclusions: The protein expression levels of p53, E-cadherin, β-catenin, MMP-10, HER2, NRAS, Met, HER2, and ERb were significantly lower in the serum collected on postoperative day 10 than in the preoperative serum, and if these proteins are consistently not elevated in the serum 3 months after surgery compared with the preoperative serum, these proteins can be potential oncogenic proteins. However, we also found that the serum extracted 3 months after the operation had elevated levels of oncogenic proteins compared with that of the preoperative and 10-day postoperative serum indicating the possibility of tumor recurrence. At postoperative follow-up period, ipsilateral neck metastasis and second primary lesion were found and additional surgery was performed to the patient. IP-HPLC using the patient's serum shows the possibility of oncogenic protein detection. However, follow-up IP-HPLC data is needed to find out patient-specific prognostic factors.

• IMMUNE NETWORK
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• 제3권4호
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• pp.276-280
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• 2003

Background: Peroxidases (Prx) of the peroxiredoxin family reduce hydrogen peroxide and alkyl hydroperoxides to water and alcohol respectively. Hydrogen peroxide is implicated as an intracellular messenger in various cellular responses such as proliferation and differentiation. And Prx I activity is regulated by Cdc-2 mediated phosphorylation. This work was undertaken to investigate the proliferation role of peroxiredoxin III as a member of Prx family in Prx III overexpressed HeLa cell line. Methods: To provide further evidence of proliferation, we selected Prx III stably expressed HeLa Tet-off cell lines. Cell proliferation was examined by using proliferation reagent WST-1 in the presence or absence of doxycycline. Prx III, 2-cys Prx enzymes exist as homodimer. The activation of Prx III heterodimer with induced and endogenous Prx III was examined by immunoprecipitation. Results: Immunoprecipitation analysis of the induced and endogenous Prx III with anti-myc showed that the induced wild type (WT) and dominant negative (DN) Prx III from HeLa Prx III Tet-off stable cell heterodimerized with endogenous Prx III each other. And the expression level of induced Prx III was examined after addition of doxycycline. By 72 hr, the expression level of induced Prx III was diminished gradually and the half-life of the induced wild type Prx III was approximately 17 hr. The proliferation experiment demonstrated that the relative proliferation value of induced and endogenous WT Prx III stable cell has no changes but the DN Prx III induced HeLa Tet-off stable cells were lower than endogenous Prx III. Conclusion: In conclusion, the HeLa dominant negative Prx III Tet-off stable cells were decreased the proliferation.

• Asian-Australasian Journal of Animal Sciences
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• 제31권1호
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• pp.3-12
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• 2018

Objective: DNA methylation plays a major role in regulating the expression of genes related to traits of economic interest (e.g., weight gain) in livestock animals. This study characterized and investigated the functional inferences of genome-wide DNA methylome in the loin (longissimus dorsi) muscle (LDM) of swine. Methods: A total of 8.99 Gb methylated DNA immunoprecipitation sequence data were obtained from LDM samples of eight Duroc pigs (four pairs of littermates). The reference pig genome was annotated with 78.5% of the raw reads. A total of 33,506 putative methylated regions (PMR) were identified from methylated regions that overlapped at least two samples. Results: Of these, only 3.1% were commonly observed in all eight samples. DNA methylation patterns between two littermates were as diverse as between unrelated individuals (p = 0.47), indicating that maternal genetic effects have little influence on the variation in DNA methylation of porcine LDM. The highest density of PMR was observed on chromosome 10. A major proportion (47.7%) of PMR was present in the repeat regions, followed by introns (21.5%). The highest conservation of PMR was found in CpG islands (12.1%). These results show an important role for DNA methylation in species- and tissue-specific regulation of gene expression. PMR were also significantly related to muscular cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism. Conclusion: This study indicated the biased distribution and functional role of DNA methylation in gene expression of porcine LDM. DNA methylation was related to cell development, cell-cell communication, cellular integrity and transport, and nutrient metabolism (e.g., insulin signaling pathways). Nutritional and environmental management may have a significant impact on the variation in DNA methylation of porcine LDM.

• 한국동물학회지
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• 제33권1호
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• pp.35-44
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• 1990

생쥐 악하선으로부터 분리한 전 RNA를 poly(U)-sepharose chromatography와 sucrose linear density gradient centrifugation 방법으로 레닌 mRNA를 분리하여 in vitro translation과 immunoprecipitation에 의하여 레닌 mRNA를 확인하였다. 레닌 mRNA로부터 레닌 cDNA를 합성하여 EcoRI inker를 이용하여 pUC19에 삽입시켰고, Taq, polymerase를 이용한 PCR방법으로 합성한 레닌 cDNA는 pUC19의 Hinalll 부의에 삽입하여 재조합 plamid를 각각 작성하였다. 이것을 JM103에 형질전환시켜 레닌 유전자 발현을 유도하여 45,000의 분자량을 갖는 레닌을 얻었으며 이 레닌 단백은 토끼으 혈압을 850115mmHg에서 115-140mmHg로 증가시키는 생리 활성을 나타냈다. 토끼의 신장 DNA를 EMBL3 phage에 삽입시켜 genomic library를 작성한 후, 레닌 cDNA로부터 합성한 probe로 plaque hybridization시켜 genomic 유전자를 갖는 재조합 phage를 분리하였다.

• Molecules and Cells
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• 제19권1호
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• pp.39-45
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• 2005

RPK118 is a sphingosine kinase-1-binding protein that has been implicated in sphingosine 1 phosphate-mediated signaling. It contains a PX (phox homology) domain and two pseudo-kinase domains, and co-localizes with sphingosine kinase-1 on early endosomes. In this study we identified a novel RPK118-binding protein, PRDX3 (peroxiredoxin-3), by yeast two-hybrid screening. The interaction between these proteins was confirmed by pull-down assays and co-immunoprecipitation experiments. Deletion studies showed that RPK118 interacted with PRDX3 through its pseudokinase domains, and with early endosomes through its PX domain. Double immunofluorescence experiments demonstrated that PRDX3 co-localized with RPK118 on early endosomes in COS7 cells. PRDX3 is a member of the antioxidant family of proteins synthesized in the cytoplasm and functioning in mitochondria. Our findings indicate that RPK118 is a PRDX3-binding protein that may be involved in transporting PRDX3 from the cytoplasm to its mitochondrial site of function or to other membrane structures via endosome trafficking.

• Journal of Microbiology and Biotechnology
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• 제31권10호
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• pp.1358-1365
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• 2021

To clarify the role of long intergenic nonprotein-coding RNA 1232 (LINC01232) in the progression of gastric cancer and the potential mechanism, we analyzed the expression of LINC01232 in TCGA database using the GEPIA online tool, and the LINC01232 level in gastric cancer cell lines was detected by quantitative real time-polymerase chain reaction (qRT-PCR) as well. Cell proliferation assay, colony formation assay, transwell assay and tumor formation experiment in nude mice were conducted to observe the biological behavior changes of gastric cancer cells through the influence of LINC01232 knockdown. LncATLAS database and subcellular isolation assay were used for subcellular distribution of LINC01232 in gastric cancer cells. The interaction among LINC01232, zeste homolog 2 (EZH2) and kruppel-like factor 2 (KLF2) was clarified by RNA-protein interaction prediction (RPISeq), RNA immunoprecipitation (RIP), qRT-PCR and chromatin immunoprecipitation (ChIP) assay. Rescue experiments were further conducted to elucidate the biological function of LINC01232/KLF2 axis in the progression of gastric cancer. LINC01232 was upregulated in stomach adenocarcinoma (STAD) tissues and gastric cancer lines. LINC01232 knockdown inhibited the proliferative capacities of gastric cancer cells in vitro, and impaired in vivo tumorigenicity. LINC01232 was mainly distributed in the cell nucleus where it epigenetically repressed KLF2 expression via binding to the enhancer of EZH2, which was capable of binding to promoter regions of KLF2 to induce histone H3 lysine 27 trimethylation (H3K27me3). LINC01232 exerts oncogenic activities in gastric cancer via inhibition of KLF2, and therefore, the knockdown of KLF2 could reverse the regulatory effect of LINC01232 in the proliferative ability of gastric cancer cells.

• 미생물학회지
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• 제54권4호
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• pp.325-329
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• 2018

PCI 영역을 포함하고 있는 Thp1/PCID2 단백질은 mRNA 전사와 방출을 연결하는, 진화적으로 보존된 TREX-2 복합체의 구성요소이다. 분열효모인 Schizosaccharomyces pombe에서 pci2 (SPBC1105.07c) 유전자는 TREX-2 복합체의 구성요소인 Thp1 (출아효모)/PCID2 (사람)의 분열효모 이종상동체로 추정되는 PCI 영역을 갖는 단백질을 암호화하고 있다. pci2 발현을 억제하면 생장과 mRNA 방출이 모두 억제되었다. 그리고 pci2 유전자의 과발현 또한 생장을 늦추고 $poly(A)^+$ RNA를 핵 안에 약간 축적되게 하였다. 뿐만 아니라 Yeast two-hybrid와 공동침전(Co-immunoprecipitation) 분석 실험에서 Pci2는 TREX-2 복합체의 다른 구성요소인 Sac3, Dss1 단백질들과 물리적으로 상호작용하였다. 이와 같은 관찰들은 S. pombe의 Pci2 단백질도 TREX-2 복합체의 구성요소로서 mRNA 방출에 관여함을 의미한다.

• BMB Reports
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• 제53권12호
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• pp.634-639
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• 2020

In prostate cancer, the androgen receptor (AR) transcription factor is a major regulator of cell proliferation and metastasis. To identify new AR regulators, we focused on Mixed lineage leukemia 5 (MLL5), a histone-regulating enzyme, because significantly higher MLL5 expression was detected in prostate cancer tissues than in matching normal tissues. When we expressed shRNAs targeting MLL5 gene in prostate cancer cell line, the growth rate and AR activity were reduced compared to those in control cells, and migration ability of the knockdown cells was reduced significantly. To determine the molecular mechanisms of MLL5 on AR activity, we proved that AR physically interacted with MLL5 and other co-factors, including SET-1 and HCF-1, using an immunoprecipitation method. The chromatin immunoprecipitation analysis showed reduced binding of MLL5, co-factors, and AR enzymes to AR target gene promoters in MLL5 shRNA-expressing cells. Histone H3K4 methylation on the AR target gene promoters was reduced, and H3K9 methylation at the same site was increased in MLL5 knockdown cells. Finally, xenograft tumor formation revealed that reduction of MLL5 in prostate cancer cells retarded tumor growth. Our results thus demonstrate the important role of MLL5 as a new epigenetic regulator of AR in prostate cancer.