• 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.

• IMMUNE NETWORK
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• 제21권4호
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• pp.30.1-30.12
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• 2021

High expression of mitofusin-2 (MFN2), a mitochondrial fusion protein, has been frequently associated with poor prognosis of patients with cervical cancer. Here, we aimed to identify the function of MFN2 in cervical cancer to understand its influence on disease prognosis. To this end, from cervical adenocarcinoma, we performed an MTT assay and quantitative RT-PCR (qRT-PCR) analysis to assess the effects of MFN2 on the proliferation and of HeLa cells. Then, colony-formation ability and tumorigenesis were evaluated using a tumor xenograft mouse model. The migration ability related to MFN2 was also measured using a wound healing assay. Consequently, epithelial-mesenchymal transition (EMT) of MFN2-knockdowned HeLa cells originating from adenocarcinoma. markers related to MFN2 were assessed by qRT-PCR. Clinical data were analyzed using cBioPortal and The Cancer Genome Atlas. We found that MFN2 knockdown reduced the proliferation, colony formation ability, migration, and in vivo tumorigenesis of HeLa cells. Primarily, migration of MFN2-knockdowned HeLa cells decreased through the suppression of EMT. Thus, we concluded that MFN2 facilitates cancer progression and in vivo tumorigenesis in HeLa cells. These findings suggest that MFN2 could be a novel target to regulate the EMT program and tumorigenic potential in HeLa cells and might serve as a therapeutic target for cervical cancer. Taken together, this study is expected to contribute to the treatment of patients with cervical cancer.

• Asian-Australasian Journal of Animal Sciences
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• 제25권10호
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• pp.1457-1465
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• 2012

Intestinal phosphate (Pi) absorption across the apical membrane of small intestinal epithelial cells is mainly mediated by the type IIb Na-coupled phosphate co-transporter (NaPi-IIb), but its expression and regulation in the chicken remain unclear. In the present study, we investigated the mRNA and protein levels of NaPi-IIb in three regions of chicken small intestine, and related their expression levels to the rate of net phosphate absorption. Our results showed that maximal phosphate absorption occurs in the jejunum, however the highest expression levels of NaPi-IIb mRNA and protein occurs in the duodenum. In response to a low-Pi diet (TP 0.2%), there is an adaptive response restricted to the duodenum, with increased brush border membrane (BBM) Na-Pi transport activity and NaPi-IIb protein and mRNA abundance. However, when switched from a low-(TP 0.2%) to a normal diet (TP 0.6%) for 4 h, there is an increase in BBM NaPi-IIb protein abundance in the jejunum, but no changes in BBM NaPi-IIb mRNA. Therefore, our study indicates that Na-Pi transport activity and NaPi-IIb protein expression are differentially regulated in the duodenum vs the jejunum in the chicken.

• Journal of Microbiology
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• 제41권4호
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• pp.295-299
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• 2003

Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1785-1790
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• 2013

The synthetic machinery of ATF4 (activating transcription factor 4) is activated in response to various stress conditions involved in nutrient restriction, endoplasmic reticulum homeostasis, and oxidation. Stress-induced inhibition of proteasome activity triggers the unfolded protein response and endoplasmic reticulum stress, where ATF4 is crucial for consequent biological events. In the current study, we showed that the $NAD^+$-dependent deacetylase, SIRT1, suppresses ATF4 synthesis during proteasome inhibition. SIRT1 depletion via transfection of specific siRNA into HeLa cells resulted in a significant increase in ATF4 protein, which was observed specifically in the presence of the proteasome inhibitor MG132. Consistent with SIRT1 depletion data, transient transfection of cells with SIRT1-overexpressing plasmid induced a decrease in the ATF4 protein level in the presence of MG132. Interestingly, however, ATF4 mRNA was not affected by SIRT1, even in the presence of MG132, indicating that SIRT1-induced suppression of ATF4 synthesis occurs under post-transcriptional control. Accordingly, we propose that SIRT1 serves as a negative regulator of ATF4 protein synthesis at the post-transcriptional level, which is observed during stress conditions, such as proteasome inhibition.

• 한국응용곤충학회지
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• 제34권4호
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• pp.287-294
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• 1995

미국흰불나방(Hyphantria cunea Drury) 번데기의 휴면 종료에 미치는 탈피호르몬 20-hydroxyecdysone (20-HE)의 처리효과와 또한 호르몬처리로 유기된 휴면 후 성충발육 모습을 정상적인 성충발육과 비교하기 위하여 본실험을 수행하였으며, 그 결과 다음고 같다. 광주기 16L.8D와 $25\pm1^{\circ}C$에서 여러가지 다른 기간동안 저장했던 휴면번데기에 20-HE를 처리했을 때 가장 높은 휴면 종료율은 가장 오랫동안 처리된 군에서 나타났고 45일 동안 저장했던 휴면번데기에서 가장 낮은 종료율을 보였다. 우화에 필요한 기간은 저온($0\pm1^{\circ}C$)처리가간에 반비례하였고, 또한 저온처리기간이 길어질 수록 20-HE에 대한 민감도는 높아졌다. 번데기 휴면기간은 그들이 16L.8D 광조건에 처리되었건 혹은 지속적이 암조건에 저장되었건 간에 거의 같았고, 또한 저온처리없이도 성공적으로 성충이 출현하였다. 정상적으로 무화(휴면없이)하는 번데기의 산소소모율은 전형적인 U자 모양을 보였지만 20-HE에 처리된 휴면 번데기의 산소흡수량은 천천히 증가하다가 처리 후 14일에 급격히 증가하였다. 번데기 휴면은 성충조직을 형성하기 이전에 들어갔으며, 정상적으로 발육하는 번데기나 또는 20-HE에 처리되어 발육하기 시작하는 휴면 번데기 모두에서 성충조직 형성시기와 대사율이 증가하기 시작하는 시기는 일치하였다. 탈피호르몬처리로 발육을 시작한 휴면 번데기의 혈림프와 지방체에서 분리된 단백질종류는 모두 정상적으로 발육하는 번데기의 조직에서와 같았다.

• 한국동물학회지
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• 제38권4호
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• pp.457-464
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• 1995

인간의 상피세포로부터 유래된 암세포의 일종인 HeLa 세포는 거의 모든 기질분자에 부착하지만 spreading은 오직 collagen 혹은 gelatin에서만 일어난다. HeLa 세포의 spreading은 오직 collagen 결과 활성화된 phosphoipase $A_2$(PLA$_2$)에 의한 arachidonic acid(AA)의 형성으로 유도된다. PLA$_2$에 의해 분해되는 인지질을 확인하기 위해 각종 인지질의 농도변화를 spreading 과정에서 측정한 결과 단지 phosphatidylcoline 만이 감소하였으며, 또한 다양한 lysophospholipids 중 lysophosphatidylcholine 만이 spreading 과정에 생성되는 것으로 보아 phosphatidylcoline이 PLA$_2$에 의해 분해되어 AA가 형성되는 것으로 보인다. PLA$_2$의 활성화는 세포질 CA$_2$+의 농도변화 및 세포질 pH의 알칼리화에 기인하지 않으며, 또한 pertussis 혹은 cholera toxin-sensitive G protein 역시 PLA$_2$활성화와는 무관한 것으로 나타났다.

• 생약학회지
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• 제48권3호
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• pp.173-178
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• 2017

We investigated the efficacy of single compound of plant extract in coxsackievirus B3 (CVB3) infection. CVB3 is a main cause of Hand-foot-mouth diseases (HFMD) and viral myocarditis in children and adult. Several single compounds of plant extract were purified by HPLC and tested as antiviral drug candidate. Among them, kaempferol was selected to effective anti-enterovirus compound by HeLa cells survival assay. CVB3 infected HeLa cells were treated with kaempferol ($100{\mu}g/ml-100ng/ml$) and their antiviral effect was confirmed. After 16 hours of treatment, HeLa cells were lysed and proteins were extracted for western blot analysis. CVB3 viral capsid protein VP1 production and transcription factor eIF4G-1 cleavage was significantly decreased in $100{\mu}g/ml$ kaempferol treatment. Virus replication was observed by virus RNA amplification. Kaempferol strongly reduced virus positive and negative strand RNA amplification. Moreover, MAPK signal induced by CVB3 infection, pERK and pmTOR, kaempferol treatment significantly inhibited the activity. Plant extract single compound, kaempferol, is a strong candidate to be developed non-toxic anti-enterovirus treatment agent.

• BMB Reports
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• 제32권4호
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• pp.379-384
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• 1999

Taxol, a natural product with significant anti-tumor activity, stabilizes microtubules and arrests cells in the G2/M phase of the cell cycle. It has been reported that taxol has additional effects on the cell such as an increase in tyrosine phosphorylation of proteins and activation of mitogen-activated protein (MAP) kinase. This phosphorylated kinase translocates into the nucleus and phosphorylates its substrate c-jun, c-fos, ATF2, and ATF3. The MAP kinase family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. First examination was cytotoxicity and apoptosis-induced concentration with paclitaxel in HeLa cell. A half-maximal inhibition of cell proliferation ($IC_{50}$) occurred at 13 nM paclitaxel. When DNA fragmentation was analyzed by agarose gel electrophoresis, a nucleosomal ladder became evident 24 h after a taxol (50 nM) addition to the cells. In addition, an apoptotic body was detected by electron microscopy. Taxol-treated cells were arrested at the S phase at 10 nM. Treatment of 50 nM taxol activated the extracellular signal-regulated protein kinase (ERK1), and a fraction of the activated MAP kinases entered the nucleus. It was also discovered that nucleus substrates c-jun was phosphorylated and activated in the cell. The activated ERK1 could subsequently translocate into the nucleus and phosphorylate its substrate c-jun as well. This study suggests that taxol-induced apoptosis might be related with signal transduction via MAP kinases.

• 생약학회지
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• 제24권4호
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• pp.267-281
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• 1993

To find antitumor components from higher fungi, the mycelia of Collybia confluens (Pers. ex Fr.) Kummer were cultured in artificial media. For efficient production of the mycelia, the influences of various modifications of culture conditions were examined. A water-soluble protein-bound polysaccharide fraction, Fr. A, was obtained from the mycelia by hot water extraction. When Fr. A was purified and fractionated by DEAE-cellulose and Sepbadex G-200 gel filtration chromatographies into four fractions which were designated B, C, C-I and C-II. The tumor inhibition ratios of these fractions ranged from 46% to 75% against the solid forms of sarcoma 180 in ICR mice at doses of 20 and 50 mg/kg/day when given intraperitoneally. Especially, Fr. C which was named Collyban(CB) exhibited a marked life-prolonging effect of the mice against ascitic forms of sarcoma 180 at a dose of 50 mg via i.p. administration. To extend spectra of the antitumor activities and eliminate the effects of allograft rejection, the characterization of antitumor effects of CB was performed in syngeneic host-tumor systems. It did not show any antitumor activity against L1210 murine leukemia in $CD_2Fl$ mice but prolonged their life span against ascitic forms of $MM_{46}$ carcinoma in $C_3H/He$ mice. Also it exhibited antitumor activity against human cervical cancer HeLa cells that were xenografted into nude mice having BALB/c genetic backgrounds by the i.p. injection at a dose of 100 mg/kg/day. In order to characterize the antitumor components, CB was examined by chemical analysis. It was acidic protein-bound polysaccharides composed of 31% polysaccharide, 27% protein and 3% hexosamine. CB was fractionated into two fractions, Fr. C-I(M.W.: 500 Kd) and Fr. C-II(M.W.:30 and 8 Kd) by Sephadex G-200 gel filtration chromatography.