• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1239-1248
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• 2004

The methylotrophic yeast Hansenula polymorpha has been extensively studied as a model organism for methanol metabolism and peroxisome biogenesis. Recently, this yeast has also attracted attention as a promising host organism for recombinant protein production. Here, we describe the fabrication and evaluation of a DNA chip spotted with 382 open reading frames (ORFs) of H. polymorpha. Each ORF was PCR-amplified using gene-specific primer sets, of which the forward primers had 5'-aminolink. The PCR products were printed in duplicate onto the aldehyde-coated slide glasses to link only the coding strands to the surface of the slide via covalent coupling between amine and aldehyde groups. With the partial genome DNA chip, we compared efficiency of direct and indirect cDNA target labeling methods, and found that the indirect method, using fluorescent-labeled dendrimers, generated a higher hybridization signal-to-noise ratio than the direct method, using cDNA targets labeled by incorporation of fluorescence-labeled nucIeotides during reverse transcription. In addition, to assess the quality of this DNA chip, we analyzed the expression profiles of H. polymorpha cells grown on different carbon sources, such as glucose and methanol, and also those of cells treated with the superoxide­generating drug, menadione. The profiles obtained showed a high-level induction of a set of ORFs involved in methanol metabolism and oxidative stress response in the presence of methanol and menadione, respectively. The results demonstrate the sensitivity and reliability of our arrays to analyze global gene expression changes of H. polymorpha under defined environmental conditions.

• Asian Pacific Journal of Cancer Prevention
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• 제15권16호
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• pp.6949-6954
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• 2014

Background: Recent studies have indicated that microRNA-15a (miR-15a) is dysregulated in breast cancer (BC). We aimed to evaluate the expression of miR-15a in BC tissues and corresponding para-carcinoma tissues. We also focused on effects of miR-15a on cellular behavior of MDA-MB-231 and expression of its target gene synuclein-${\gamma}$ (SNCG). Materials and Methods: The expression levels of miR-15a were analysed in BC formalin fixed paraffin embedded (FFPE) tissues by microarray and quantitative real-time PCR. CCK-8 assays, cell cycle and apoptosis assays were used to explore the potential functions of miR-15a in MDA-MB-231 human BC cells. A luciferase reporter assay confirmed direct targets. Results: Downregulation of miR-15a was detected in most primary BCs. Ectopic expression of miR-15a promoted proliferation and suppressed apoptosis in vivo. Further studies indicated that miR-15a may directly interact with the 3'-untranslated region (3'-UTR) of SNCG mRNA, downregulating its mRNA and protein expression levels. SNCG expression was negatively correlated with miR-15a expression. Conclusions: MiR-15a has a critical role in mediating cell cycle arrest and promoting cell apoptosis of BC, probably by directly targeting SNCG. Thus, it may be involved in development and progression of BC.

• Journal of Acupuncture Research
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• 제37권2호
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• pp.128-135
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• 2020

Background: Classical acupuncture is being used in the treatment of rheumatoid arthritis (RA). To explore the biological response to acupuncture, a network-based analysis was performed on gene expression data collected from an animal model of RA treated with acupuncture. Methods: Gene expression data were obtained from published microarray studies on blood samples from rats with collagen induced arthritis (CIA) and non-CIA rats, both treated with manual acupuncture. The weighted gene co-expression network analysis was performed to identify gene clusters expressed in association with acupuncture treatment time and RA status. Gene ontology and pathway analyses were applied for functional annotation and network visualization. Results: A cluster of 347 genes were identified that differentially downregulated expression in association with acupuncture treatment over time; specifically in rats with CIA with module-RA correlation at 1 hour after acupuncture (-0.27; p < 0.001) and at 34 days after acupuncture (-0.33; p < 0.001). Functional annotation showed highly significant enrichment of porphyrin-containing compound biosynthetic processes (p < 0.001). The network-based analysis also identified a module of 140 genes differentially expressed between CIA and non-CIA in rats (p < 0.001). This cluster of genes was enriched for antigen processing and presentation of exogenous peptide antigen (p < 0.001). Other functional gene clusters previously reported in earlier studies were also observed. Conclusion: The identified gene expression networks and their hub-genes could help with the understanding of mechanisms involved in the pathogenesis of RA, as well understanding the effects of acupuncture treatment of RA.

• 원예과학기술지
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• 제33권4호
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• pp.575-584
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• 2015

건조 스트레스는 작물의 생존과 생산성을 결정하는데 매우 중요한 환경요인이다. 본 연구의 목적은 배추에서 신규 건조 스트레스 저항성 유전자를 동정 검정하는 것이다. 건조 스트레스 하에서 생육된 지부('Chiifu') 배추를 이용하여 제작된 KBGP-24K 마이크로어레이 데이터 분석을 통해 738개의 건조 반응 유전자 중 기능은 밝혀져 있지 않지만, 건조 스트레스 하에서 발현량이 6배 이상 크게 증가한 1개의 유전자를 선발하여 BrDSR(B. rapa Drought Stress Resistance)이라 명명하였다. 이의 검정을 위해 내혼계배추('CT001')에서 BrDSR을 동정한 결과 438bp의 오픈리딩프레임과 145개의 아미노산을 가지고 있음을 확인하였고, 동정된 완전장의 cDNA 염기서열은 형질전환용 과발현 vector인 'pSL100' 제작에 이용하였다. BrDSR이 식물체에서 건조 스트레스 저항성을 향상시켜줄 수 있는지 분석하기 위해 담배 형질전환을 수행하였다. PCR과 DNA 블롯 분석으로 선발된 T1 세대 담배 형질전환체들을 대상으로 quantitative real-time RT PCR 분석을 수행한 결과, 형질전환체의 BrDSR 발현량은 비형질 전환체 보다 2.6배까지 증가하였다. 또한 건조처리 10일째 수행한 표현형 분석에서 BrDSR이 발현되는 담배 형질전환체들이 비형질전환체들 보다 우수한 건조 저항성을 보였다. 연구 결과들을 종합하면 BrDSR은 건조 스트레스 하에서 식물의 생장과 생존에 효과적인 저항성 기능을 할 것으로 기대된다.

• Animal cells and systems
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• 제14권4호
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• pp.275-282
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• 2010

Chunghyul-dan (CHD) is a combinatorial drug known to exert anti-inflammatory effects in endothelial cells. In this study, we employed global transcriptional profiling using cDNA microarrays to identify molecular mechanisms responsible for the anti-inflammatory activity of CHD in endothelial cells. An analysis of the microarray data revealed that transcript levels of monocyte chemotactic protein-1 (MCP-1), vascular cell-adhesion molecule-1 (VCAM-1) and activated leukocyte cell-adhesion molecule were dramatically altered in CHD-treated endothelial cells. These changes in gene expression were confirmed by RT-PCR, Western blotting and ELISA. Chronic CHD treatment also appeared to decrease MCP-1 secretion, probably as a result of decreased MCP-1 expression. In addition, we determined that chronic CHD treatment inhibited lipopolysaccharide-stimulated adhesion of THP-1 leukocytes to endothelial cells. The inhibitory effect of CHD on LPS-stimulated adhesion resulted from downregulation of VCAM-1 expression. Transmigration of THP-1 leukocytes through endothelial cells was also inhibited by chronic CHD treatment. In conclusion, CHD controls a variety of inflammatory activities by regulating MCP-1 and VCAM-1 gene expression.

• Molecules and Cells
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• 제40권2호
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• pp.123-132
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• 2017

Insulin signaling is coordinated by insulin receptor substrates (IRSs). Many insulin responses, especially for blood glucose metabolism, are mediated primarily through Irs-1 and Irs-2. Irs-1 knockout mice show growth retardation and insulin signaling defects, which can be compensated by other IRSs in vivo; however, the underlying mechanism is not clear. Here, we presented an Irs-1 truncated mutated mouse ($Irs-1^{-/-}$) with growth retardation and subcutaneous adipocyte atrophy. $Irs-1^{-/-}$ mice exhibited mild insulin resistance, as demonstrated by the insulin tolerance test. Phosphatidylinositol 3-kinase (PI3K) activity and phosphorylated Protein Kinase B (PKB/AKT) expression were elevated in liver, skeletal muscle, and subcutaneous adipocytes in Irs-1 deficiency. In addition, the expression of IRS-2 and its phosphorylated version were clearly elevated in liver and skeletal muscle. With miRNA microarray analysis, we found miR-33 was down-regulated in bone marrow stromal cells (BMSCs) of $Irs-1^{-/-}$ mice, while its target gene Irs-2 was up-regulated in vitro studies. In addition, miR-33 was down-regulated in the presence of Irs-1 and which was up-regulated in fasting status. What's more, miR-33 restored its expression in re-feeding status. Meanwhile, miR-33 levels decreased and Irs-2 levels increased in liver, skeletal muscle, and subcutaneous adipocytes of $Irs-1^{-/-}$ mice. In primary cultured liver cells transfected with an miR-33 inhibitor, the expression of IRS-2, PI3K, and phosphorylated-AKT (p-AKT) increased while the opposite results were observed in the presence of an miR-33 mimic. Therefore, decreased miR-33 levels can up-regulate IRS-2 expression, which appears to compensate for the defects of the insulin signaling pathway in Irs-1 deficient mice.

• 생명과학회지
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• 제28권12호
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• pp.1406-1415
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• 2018

본 연구는 어성초(Houttuynia cordata Thunb)의 메탄올 추출에 의한 유기 용매별 분획물에서 항산화 효과를 근거로 산화적 스트레스에 의한 HaCaT 세포보호 효과를 확인하였다. 용매별 분획물의 항산화 활성은 시료의 농도가 증가할수록 DPPH에 대한 전자공여능도 증가하였으며, $ED_{50}$은 ethyl acetate (EtOAc) 분획물에서 $175{\mu}g/ml$로 가장 높게 나타났다. $H_2O_2$에 의해 유도된 HaCaT 세포의 세포사멸($IC_{50}$)에 대하여 Hc-EtOAc 분획물은 농도 의존적으로 유의적인 세포 생존율과, $100{\mu}g/ml$ 농도에서 74%의 세포보호 효과를 나타내었다. 한편 $100{\mu}g/ml$의 Hc-EtOAc 분획물을 6 및 24시간 동안 HaCaT 세포에 처리하여 유전자 발현 양상을 분석하였다. 2 배 이상 발현이 증가된 유전자들은 신호전달, 세포분열, 항산화 활성, 상피세포 증식 등에 작용하는 것으로 나타났다. 특히 항산화 활성에 관여할 것으로 추정되는 유전자는 전염증성 사이토카인인 IL1B, TNF, 그리고 IL6 등 이었으며, 이들 유전자의 상위 조절자로써 TLR4가 확인되었다. IL1B, TNF, 그리고 IL6 유전자의 활성을 검증하기 위하여 qRT-PCR을 수행한 결과, $100{\mu}g/ml$ 이상의 Hc-EtOAc 분획물 처리군에서 2 배 이상 발현이 증가한 것으로 나타났다. 상위 조절자 TLR4 단백질의 활성 역시 Hc-EtOAc 분획물에 의해 증가되었다. 이상의 결과, Hc-EtOAc 분획물에 의한 항산화 활성은 TLR4로부터 IL1B, TNF, IL6과 같은 사이토카인을 경유하는 것으로 예측된다.