• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.946-951
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• 2006

To study the effect of iron on Saccharomyces cerevisiae, whole-cell proteins of Saccharomyces cerevisiae were extracted and subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and differentially expressed proteins were identified. The proteins separated were further identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and were compared with a protein database. Of more than 300 spots separated by molecular weight and isoelectric points, 27 differentially expressed spots were identified. Ten proteins were found to be differentially expressed at high iron concentration. Triosephosphate isomerase (TPI), YDR533C hypothetical protein, superoxide dismutase (SOD), 60 kDa heat-shock protein (HSP60), pyruvate dehydrogenase beta subunit 1 (PDB1), and old yellow enzyme 2 (OYE2) were upregulated, whereas thiol-specific antioxidant (TSA), regulatory particle non-ATPase subunit 8 (RPN8), thiol-specific peroxiredoxin 1 (AHP1), and fructose-1, 6-bisphosphate adolase (FBA) were downregulated by iron. Based on the result, we propose that SOD upregulated by iron would protect the yeast from oxidative stress by iron, and that TSA downregulated by iron would render cells hypersensitive to oxidative stress.

• 한국가축번식학회지
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• 제25권1호
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• pp.71-77
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• 2001

본 실험은 phospholipase C (PLC)-$\beta$-3 및 peroxiredoxin (Prx) II 유전자가 적중 ($\Delta$)된 J1 마우스 배아주 (embryonic stem) 세포로부터 생식선이행 카이미라 마우스생산을 위한 제반조건 및 배아주세포의 배양조건을 확립하기 위하여 수행되었다. 80% 이상의 정상핵형을 보이는 유전자 적중된 4개의 클론 ($\Delta$Pu II C3, $\Delta$Pu II C3, C10 및 15)으로부터 카이미라를 생산하였을 때 형태적으로 분화정도가 높은 클론 ($\Delta$PLC$\beta$-3 C3)의 이용은 카이미라의 생산율 (21.1%)과는 무관하게 카이미리즘 (＜ 20%)이 낮았고, 수컷 카이미라의 생산 빈도 (7/15, 46%)도 낮아지는 것으로 나타났다. 그러나 형태적으로 안정된 3개의 클론 ($\Delta$Prx II C3, C10 및 15)은 80% 이상의 높은 카이미리즘을 지닌 마우스 (9/13, 69.2%)를 생산하였고, 수컷 카이미라의 생산율 (l1/13, 84.6%)도 증대된 것으로 나타났다. 따라서 80% 이상의 정상핵형을 지닌 배아주 세포를 형태적으로 안정하게 유지하는 것이 카이미리즘이 높은 마우스를 생산하는데 결정적 요인으로 작용할 수 있는 것으로 확인되었다. 미세주입용 배반포를 효율적으로 생산하기 위해 5~10주령 사이의 C57BL/6J 암컷마우스를 교배 한 결과, 10주령 마우스가 미세주입가능한 3.5일령 배반포를 가장 많이 생산 (2.94개/마리)하였다. 또한 미세주입된 배반포를 이식하기 위해 ICR 및 ICR$\times$C57BL/6J F1 (IBF1)위임신 대리모를 사용하였을 때, IBF1이 복당 산자수 (2.8 vs. 5.6)가 많았고 카이미라 생산율 (0 vs. 35.3%)도 매우 높았다. 따라서 공여마우스의 주령 및 대리모의 선택이 카이미라 생산효율 향상에 중요한 요인으로 부각되었다. 결과적으로 핵형이 안정된 ES 세포를 동정하는 것은 물론 클로닝 과정중에 형태적으로 분화가 없도록 ES세포를 배양하는 것이 카이미리즘이 높은 마우스를 생산하고 아울러 생식선 이행 빈도를 증가시키는데 결정적인 역할을 하는 것으로 확인되었다.

• Journal of Korean Neurosurgical Society
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• 제59권6호
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• pp.544-550
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• 2016

Objective : Cerebral endothelial cells have unique biological features and are fascinating candidate cells for stroke therapy. Methods : In order to understand the molecular mechanisms of human cerebral endothelial cell (hCMEC/D3) transplantation in a rat stroke model, we performed proteomic analysis using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein expression was confirmed by quantitative real-time PCR and Western blot. Results : Several protein spots were identified by gel electrophoresis in the sham, cerebral ischemia (CI), and CI with hCMEC/D3 treatment cerebral ischemia with cell transplantation (CT) groups, and we identified 14 differentially expressed proteins in the CT group. Proteins involved in mitochondrial dysfunction (paraplegin matrix AAA peptidase subunit, SPG7), neuroinflammation (peroxiredoxin 6, PRDX6), and neuronal death (zinc finger protein 90, ZFP90) were markedly reduced in the CT group compared with the CI group. The expression of chloride intracellular channel 4 proteins involved in post-ischemic vasculogenesis was significantly decreased in the CI group but comparable to sham in the CT group. Conclusion : These results contribute to our understanding of the early phase processes that follow cerebral endothelial cell treatment in CI. Moreover, some of the identified proteins may present promising new targets for stroke therapy.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
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• pp.137-137
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• 2005

• Clinical and Experimental Reproductive Medicine
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• 제38권1호
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• pp.18-23
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• 2011

Objective: Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. This report examined the expression of Prx isotype I in the rat ovary after hormone treatment. Methods: Immature rats were injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) to induce the growth of multiple preovulatory follicles and 10 IU of human chorionic gonadotropin (hCG) to induce ovulation. Immature rats were also treated with diethylstilbestrol (DES), an estrogen analogue, to induce the growth of multiple immature follicles. Northern blot analysis was performed to detect gene expression. Cell-type specific localization of Prx I mRNA were detected by in situ hybridization analysis. Results: During follicle development, ovarian Prx I gene expression was detected in 3-day-old rats and had increased in 21-day-old rats. The levels of Prx I mRNA slightly declined one to two days following treatment with DES. A gradual increase in Prx I gene expression was observed in ovaries obtained from PMSG-treated immature rats. Furthermore, hCG treatment of PMSG-primed rats resulted in a gradual stimulation of Prx I mRNA levels by 24 hours (2.1-fold increase) following treatment, which remained high until 72 hours following treatment. In situ hybridization analysis revealed the expression of the Prx I gene in the granulosa cells of PMSG-primed ovaries and in the corpora lutea of ovaries stimulated with hCG for 72 hours. Conclusion: These results demonstrate the gonadotropin and granulosa cell-specific stimulation of Prx I gene expression, suggesting its role as a local regulator of follicle development.

• 한국환경생태학회지
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• 제30권5호
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• pp.810-820
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• 2016

염생식물 퉁퉁마디의 종자 발아에 영향을 미치는 환경 요인을 조사하고 환경 스트레스에 의해 유도되는 2CysPrx 유전자를 클로닝한 후 스트레스 조건에 따른 2CysPrx 유전자의 발현 양상에 대하여 조사하였다. 염생식물에 대한 가장 대표적인 스트레스는 염분 스트레스로서 퉁퉁마디 발아에 중요한 요인으로 작용하고 있다. 퉁퉁마디의 발아에 대한 NaCl의 한계 농도는 7%로 나타났고, 최적의 발아 조건은 NaCl이 없는 상태로 확인되었다. 퉁퉁마디 발아에서 최적 온도는 $20^{\circ}C$로 98%의 발아율을 보였다. 스트레스에 유도되는 유전자 후보군 중 2CysPrx 유전자의 cDNA를 클론하여 분석한 결과 275개의 아미노산으로 이루어져 있고 두 개의 시스테인 잔기를 가지고 있으며 분자량은 30.1kDa으로 나타났다. 2CysPrx 유전자는 서던 블롯에 의해 유전체에 한 카피 존재하는 것으로 나타났고, 6개의 인트론과 7개의 엑손으로 구성되어 있다. qPCR에 의한 2CysPrx 유전자의 전사율을 분석한 결과, 3.5% NaCl과 40mM $H_2O_2$ 처리 조건에서 전사율이 가장 높게 나타났고, 고온($40^{\circ}C$)과 $75{\mu}M$ ABA 처리 조건에서는 처리 후 8시간에 최고의 전사율을 보였으며, 저온($4^{\circ}C$)에서는 유전자 발현이 일어나지 않는 것으로 나타났다. 우리는 여러 환경 스트레스에 의해 유도되는 다른 유전자의 클로닝을 시도하고 있다.

• Journal of Ginseng Research
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• 제40권2호
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• pp.185-195
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• 2016

Background: To investigate the effect of pectinase-treated Panax ginseng (GINST) in cellular and male subfertility animal models. Methods: Hydrogen peroxide ($H_2O_2$)-induced mouse spermatocyte GC-2spd cells were used as an in vitro model. Cell viability was measured using MTT assay. For the in vivo study, GINST (200 mg/kg) mixed with a regular pellet diet was administered orally for 4 mo, and the changes in the mRNA and protein expression level of antioxidative and spermatogenic genes in young and aged control rats were compared using real-time reverse transcription polymerase chain reaction and western blotting. Results: GINST treatment ($50{\mu}g/mL$, $100{\mu}g/mL$, and $200{\mu}g/mL$) significantly (p < 0.05) inhibited the $H_2O_2$-induced ($200{\mu}M$) cytotoxicity in GC-2spd cells. Furthermore, GINST ($50{\mu}g/mL$ and $100{\mu}g/mL$) significantly (p < 0.05) ameliorated the $H_2O_2$-induced decrease in the expression level of antioxidant enzymes (peroxiredoxin 3 and 4, glutathione S-transferase m5, and glutathione peroxidase 4), spermatogenesis-related protein such as inhibin-${\alpha}$, and specific sex hormone receptors (androgen receptor, luteinizing hormone receptor, and follicle-stimulating hormone receptor) in GC-2spd cells. Similarly, the altered expression level of the above mentioned genes and of spermatogenesis-related nectin-2 and cAMP response element-binding protein in aged rat testes was ameliorated with GINST (200 mg/kg) treatment. Taken together, GINST attenuated $H_2O_2$-induced oxidative stress in GC-2 cells and modulated the expression of antioxidant-related genes and of spermatogenic-related proteins and sex hormone receptors in aged rats. Conclusion: GINST may be a potential natural agent for the protection against or treatment of oxidative stress-induced male subfertility and aging-induced male subfertility.

• International Journal of Oral Biology
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• 제34권4호
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• pp.205-213
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• 2009

The mechanisms underlying the actions of the antioxidants upon reactive oxygen species (ROS) generation by NADPH oxidase complex have remained uncertain. In this study, we investigated NADPH oxidase activity and the role of antioxidant enzymes upon the generation of ROS during hypoxic stress. ROS generation was found to increase in the mouse kidney under hypoxic stress in a time-dependent manner. Moreover, we found in MCT cells that hypoxia-induced hydrogen peroxide production was decreased by NAC pretreatment. We further analyzed HIF-$1{\alpha}$, PHD2 and VHL expression in the NAC-pretreated MCT cells and assessed the response of antioxidant enzymes at the transcriptional and translational levels. SOD3 and Prdx2 were significantly increased during hypoxia in the mouse kidney. We also confirmed in hypoxic $Prdx2^{-/-}$ and SOD3 transgenic mice that erythropoietin (EPO) is transcriptionally regulated by HIF-$1{\alpha}$. In addition, although EPO protein was found to be expressed in a HIF-$1{\alpha}$ independent manner in three mouse lines, its activity differed markedly between normal and $Prdx2^{-/-}$/SOD3 transgenic mice during hypoxic stress. In conclusion, our current results indicate that NADPH oxidase-mediated ROS generation is associated with hypoxic stress in the mouse kidney and that SOD3 and Prdx2 cooperate to regulate cellular redox reactions during hypoxia.

• Animal Bioscience
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• 제34권4호
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• 대한한방내과학회지
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• 제32권1호
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• pp.33-42
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• 2011

Background : Despite recent advances in cancer management, prognosis of ovarian cancer is poor. Anticancer effects of herbal medicine, such as Euonymus alatus Sieb, Oldenlandia diffusa (Willd.) Roxburgh, and Orostachys japonicus A. Berger, have been reported in treatment of ovarian and cervical cancers, but the systematic approaches to explain their molecular mechanism(s) have not yet been established. Objectives : To establish a basis of understanding for anti-cancer mechanisms of herbal medicine, we profiled protein expression in human ovarian and cervical cancer cells treated with the extracts from Euonymus alatus Sieb, Oldenlandia diffusa (Willd.) Roxburgh and Orostachys japonicus A. Berger. Methods : Human ovarian cancer cell line NIH:OVCAR-3, and human cervical cancer cell line HeLa were employed in the present study. Whole protein was obtained from the cells harvested at 48 hours after the treatment with herbal water-extract, and analyzed by 2DE-based proteomic approach. Results : Various changes of protein expression induced by the herbal treatment were monitored : down-regulation of molecular chaperone (calreticulin variant), glycolytic enzymes (D-3-phosphoglycerate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase and alpha-enolase), RNA processing molecules (hnRNP A2/B1), and antioxidant protein (peroxiredoxin 1). Conclusions : Repression of glycolysis has been accepted as the mechanism to increase anticancer reagent's effect. Thus, down-regulation of glycolytic enzymes by the herbal extracts suggested a possible synergistic effect of herbs in the presence of platinum-based therapeutics. In further study, as well as the synergistic effect of the herbs, it has to be further validated whether artificial regulation of hnRNP A2/B1 in ovarian cancer cells affects various cancer survival factors, since RNA processing can be interrupted by deranged expression of hnRNP subtypes, and it results in an inhibition of cancer cell growth.