• Molecules and Cells
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• v.43 no.11
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• Archives of Pharmacal Research
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• v.21 no.5
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• pp.537-542
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• 1998

to more effectively drive immune responses toward antigen-specific T helper type 2 (Th2) cell-mediated responses, we constructed a mammalian expression vetor (oPVA/IL4) carrying a fused gene in which the ovalbumin (OVA) cDNA was covalently linked to murine interleukin-4 (IL-4) cDNA. A biologically active OVA/IL4 DNA, as demonstrated by Wes tern blotting and cytokine bioassay. In tramuscular injection of BALB/c mice with the pOVA/IL4 DNA increased both the production of OVA-specific IL-4 by CD$4^{+}$ T cells and the ratio of anti-OVA lgG1 to anti-OVA lgG2a isotypes, while the injection with the pOVA DNA alone, or with the mixture of the pOVA and pIL4 DNA did no or little increase. furthermore, the OVA-specific, Th2 cell-mediated immune responses were significantly enhanced by multiple injections with the pOVA/IL4 DNA. These studies indicate that the direct linkage of an OVA gene to an IL-4 gene in the expression plasmid confines the effects of IL-4 to the OVA-specific cells, efficiently driving the immune response toward OVA-specific, Th2 cell-mediated responses.

• Molecules and Cells
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• v.22 no.1
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• pp.58-64
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• 2006

WRKY family proteins are a class of plant-specific transcription factors involved in stress response signaling pathways. In this study a gene encoding a putative WRKY protein was isolated from a pepper EST database (http://genepool.kribb.re.kr). The cDNA, named Capsicum annuum WRKY2 (CaWRKY2), encodes a putative polypeptide of 548 amino acids, containing two WRKY domains with zinc finger motifs and two potential nuclear localization signals. Northern blot analyses showed that CaWRKY2 mRNA was preferentially induced during incompatible interactions of pepper plants with PMMoV, Pseudomonas syringae pv. syringae 61, and Xanthomonas axonopodis pv. vesicatoria race 3. Furthermore, CaWRKY2 transcripts were strongly induced by wounding and ethephon treatment, whereas only moderate expression was detected following treatment with salicylic acid and jasmonic acid. CaWRKY2 was translocated to the nucleus when a CaWRKY2-smGFP fusion construct was expressed in onion epidermal cells. CaWRKY2 also had transcriptional activation activity in yeast. Taken together our data suggest that CaWRKY2 is a pathogen-inducible transcription factor that may have a role in early defense responses to biotic and abiotic stresses.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.16-22
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• 2003

Background: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. Methods: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. Results: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. Conclusion: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis.

• BMB Reports
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• v.43 no.5
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• pp.375-381
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• 2010

In this study, the cDNA library of Chang-liver cells was immunoscreened using common ADAMs antibody to obtain ADAM related genes. We found one positive clone that was confirmed as a new gene by Blast, which is an uncharacterized helical and coil protein and processes protease activity, and named protease-related protein 1 (ARP1). The submitted GenBank accession number is AY078070. Molecular characterizations of ARP1 were analyzed with appropriate bioinformatics software. To analyse its expression and function, ARP1 was subcloned into glutathione S-transferase fusion plasmid pGEX-2T and expressed by E. coli system. The in vitro expression product of ARP1 was recognized by common ADAMs antibody with western blot. Interestingly, ARP1 cleaves gelatine at pH9.5, which suggests it is an alkaline protease. Semi-quantitative RT-PCR result indicates that ARP1 mRNA is strongly transcribed in the liver and the treated Chang-liver cells.

• Animal cells and systems
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• v.6 no.3
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• pp.271-275
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• 2002

Rph1 and Gisl are damage-responsive repressors involved in PHR1 expression. They have two $C_{2}$H/ sub 2/ zinc finger motifs as putative DNA binding domains and N-terminal conserved domain with unknown function. They are also found in the human retinoblastoma binding protein 2 and the mouse jumonji- encoded protein. The repressors are able to bind to A $G_{4}$ sequence within a 39-bp sequence called upstream repressing sequence of PHR1 promoter (UR $S_{PHR1}$) responsible for the damage-response of PHR1. We report here that Rph1 is predominantly localized in the nucleus as examined by fluorescence microscopic analysis with GFP-Rph1 fusion protein. On the basis of the fact that the A $G_{4}$ sequence that is recognized by Rph1 and Gisl is also recognized by Msn2 and Msn4 in a process of stress response, we a1so tried to examine the in vivo function of A $G_{4}$ and the role of Msn2 and Msn4 in PHR1 expression. Our results demonstrate that Msn2 and Msn4 are actually required for the basal transcription of PHR1 expression but not for its damage induction. When A $G_{4}$ sequence was inserted into the minimal promoter of the cyc1-LacZ reporter, the increased LacZ expression was observed indicating its involvement in transcriptional activation. The data suggest that the A $G_{4}$ is primarily required for basal transcriptional activation of PHR1 or CYC1 promoter through the possible involvement of Msn2 and Msn4. However, since the A $G_{4}$ is also involved in the repression of PHR1 via Rphl and Gisl, it is proposed that A $G_{4}$ functions as either URS or upstream activating sequence (UAS) depending on the promoter context.t.

• Proceedings of the Korean Vacuum Society Conference
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• 2012.02a
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• pp.156-157
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• 2012

단일 셀에서 비휘발성 및 고속의 휘발성 메모리를 모두 구동할 수 있는 다기능 메모리는 모바일 기기 및 embedded 장치의 폭발적인 성장에 있어 그 중요성이 커지고 있다. 따라서 최근 이러한 fusion기술을 응용한 unified RAM (URAM)과 같은 다기능 메모리의 연구가 주목 받고 있다. 이러한 다목적 메모리는 주로 silicon on insulator (SOI)기반의 1T-DRAM과 SONOS기술 기반의 비휘발성 메모리의 조합으로 이루어진다. 하지만 이런 다기능 메모리는 주로 단결정기반의 SOI wafer 위에서 구현되기 때문에 값이 비싸고 사용범위도 제한되어 있다. 따라서 이러한 다기능메모리를 다결정 실리콘을 이용하여 제작한다면 기판에 자유롭게 메모리 적용이 가능하고 추후 3차원 적층형 소자의 구현도 가능하기 때문에 다결정실리콘 기반의 메모리 구현은 필수적이라고 할 수 있겠다. 본 연구에서는 다결정실리콘을 이용한 channel recessed구조의 다기능메모리를 제작하였으며 각 1T-DRAM 및 NVM동작에 따른 memory 특성을 살펴보았다. 실험에 사용된 기판은 상부 비정질실리콘 100 nm, 매몰산화층 200 nm의 SOI구조의 기판을 이용하였으며 고상결정화 방법을 이용하여 $600^{\circ}C$ 24시간 열처리를 통해 결정화 시켰다. N+ poly Si을 이용하여 source/drain을 제작하였으며 RIE시스템을 이용하여 recessed channel을 형성하였다. 상부 ONO게이트 절연막은 rf sputter를 이용하여 각각 5/10/5 nm 증착하였다. $950^{\circ}C$ N2/O2 분위기에서 30초간 급속열처리를 진행하여 source/drain을 활성화 하였다. 계면상태 개선을 위해 $450^{\circ}C$ 2% H2/N2 분위기에서 30분간 열처리를 진행하였다. 제작된 Poly Si MFM에서 2.3V, 350mV/dec의 문턱전압과 subthreshold swing을 확인할 수 있었다. Nonvolatile memory mode는 FN tunneling, high-speed 1T-DRAM mode에서는 impact ionization을 이용하여 쓰기/소거 작업을 실시하였다. NVM 모드의 경우 약 2V의 memory window를 확보할 수 있었으며 $85^{\circ}C$에서의 retention 측정시에도 10년 후 약 0.9V의 memory window를 확보할 수 있었다. 1T-DRAM 모드의 경우에는 약 $30{\mu}s$의 retention과 $5{\mu}A$의 sensing margin을 확보할 수 있었다. 차후 engineered tunnel barrier기술이나 엑시머레이저를 이용한 결정화 방법을 적용한다면 device의 특성향상을 기대할 수 있을 것이다. 본 논문에서는 다결정실리콘을 이용한 다기능메모리를 제작 및 메모리 특성을 평가하였다. 제작된 소자의 단일 셀 내에서 NVM동작과 1T-DRAM동작이 모두 가능한 것을 확인할 수 있었다. 다결정실리콘의 특성상 단결정 SOI기반의 다기능 메모리에 비해 낮은 특성을 보여주었으나 이는 결정화방법, high-k절연막 적용 및 engineered tunnel barrier를 적용함으로써 해결 가능하다고 생각된다. 또한 sputter를 이용하여 저온증착된 O/N/O layer에서의 P/E특성을 확인함으로써 glass위에서의 MFM구현의 가능성도 확인할 수 있었으며, 차후 system on panel (SOP)적용도 가능할 것이라고 생각된다.

• Journal of the Korean Association of Geographic Information Studies
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• v.18 no.1
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• pp.147-155
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• 2015

The existing field survey and aerial photography involve the waste of manpower and economic loss in the coastline survey. To minimize these disadvantages, the digitization for efficient coastline extraction was conducted in this study using the points extracted from the standard coastline of the approximate highest high water and the diverse satellite images (KOMPSAT-3, SPOT-5, Landsat-8 and Quickbird-2), and the comparative accuracy analysis was conducted. The differences between the standard coastline points of the approximate highest high water and the coastline of each satellite were smallest for KOMPSAT-3, followed by Quickbird-2, SPOT-5 and Landsat-8. The significant probability from between the multipurpose applications satellite and Quickbird-2 (significant probability two-tailed) was statistically significant at 1% significance level. Therefore, high-resolution satellite images are required to efficiently extract the coastline, and KOMPSAT-3, from which images are easily acquired at a low cost, will enable the most efficient coastline extraction without external support.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1641-1646
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• 2004

The objective of this study was to generate transgenic mice expressing human resistin gene by using the tetraploidembryonic stem (ES) cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR, cloned into $pCR^{(R)}$ 2.1 $TOPO^{(R)}$ vector and constructed in pCMV-Tag4C vector. Mammalian expression plasmid containing human resistin was transfected into D3-GL ES cells by Lipofectamine 2,000, and then after 10-12 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec (fusion rate: 2,114/2,256, 93.5%) and cultured up to the blastocyst stage (development rate: 1,862/2,114, 94.6%). The selected 15-20 ES cells were injected into tetraploid blastocysts, and then transferred into the uteri of E 2.5 d pseudopregnant recipient mice. To investigate the gestation progress, two E 19.5 mused fetuses were recovered by Cesarean section of which one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, our findings demonstrate that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mice for the rapid analysis of gene function in vivo.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.5
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• pp.779-784
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• 1995

The effective production of 5'-GMP(5'-Guanylic acid) by immobilized 5'-GMP producing fusant RC102(intergeneric protoplast fusion between Brevibacterium ammoniagenes ATCC21263 and Corynebacterium glutamicum ATCC21171) was investigated. The Fusant RC102 was immobilized by entrapping in -carrageenan, agar, polyacrylamide or Ca-alginate. 3% k-carrageenan was selected as the most suitable matrix. In the production of 5'-GMP using the immobilized whole cells of fusant RC102, the optimum conditions were $32^{\circ}C$, pH 8.0, $30\mu\textrm{g}/L\;of\;Mn^{2+},\;1{\times}10^{-6}%\;of\;Zn^{2+}$. In order to use fermentation medium containing CSL(Corn Steep Liquor) plentiful in $Mn^{2+}$, the optimum conditions of penicillin G, D-cycloserine and POESA(polyoxyethylene stearylamine) for production of 5'-GMP were 0.8unit/ml, 0.8unit/ml, 0.8unit/ml and 5mg/ml, respectively. Cationic surfactant, POESA was effective and superior to the antibiotics, penicillin G or D-cyloserine in 5'-GMP productivity. The condinuous fermentation using immobilized fusant RC102 showed that 5'-GMP productivity was stable for more than 15 days.