• Animal cells and systems
• /
• v.1 no.3
• /
• pp.521-526
• /
• 1997

We isolated a mouse nck cDNA from the thymus cDNA expression library. The cDNA encodes a 377 amino acid protein and displays 97% amino acid sequence identity to human oncogenic protein nck, which is composed almost exclusivelv of three src homology 3 (SH3) domains and one SH2 domain. The sequence analysis also showed that the isolated cDNA is the mouse counterpart of the human nck and different from the mouse grb4, which has been reported to be highly similar to the human nck and, therefore considered as a mouse nck, Northern blot analysis showed that the transcript of the gene was 1.8 kb and was highly expressed in the testis, thymus, and brain but moderately in the liver and lymph node. Western blot analysis showed that the size of the protein was about 47 kDa. Overexpression of the mouse Nck transformed a mouse fibroblast cell line, NIH3T3. The results clearly indicate that normal nck gene has transforming ability and provide an argument against a suggested possibility that the transforming ability of the human nck gene is due to a mutation(s) in the gene.

• Archives of Pharmacal Research
• /
• v.20 no.5
• /
• pp.404-409
• /
• 1997

Trout CYP1A-CAT expression construct was generated by cloning -3.5 Kb $5^I$ flanking DNA of trout liver CYP1A gene in front of CAT gene at pCAT-basic vector. Hepa 1 cells, which are known to contain a functional arylhydrbcarbon $receptor^I$ were transfected with trout CYP1A-CAT using lipofectin. 3-Methylcholanthrene (1 nM) was added into hepa 1 cells in culture in order to examine if $5^I$ flanking DNA of trout CYP1A gene could interact with mouse transactivating factors to bring about transcription of the chloramphenicol acetyltransferase(CAT) reporter gene. The level of CAT protein was measured by CAT ELISA and the level of CAT mRNA was determined by RTPCR. The treatment of 1 nM 3-methylcholanthrene resulted in two fold increases in CAT protein as well as CAT mRNA compared to untreated control hepa 1 cells. These data indicate that arylhydrocarbon receptors of mouse hepa 1 cells are functional to activate exogenously transfected trout CYP1A-CAT construct in terms of both transcription and translation of CAT. We also examined the effect of 3-methylcholanthrene on endogenous cyplal activity in hepa 1 cell. 3-Methylcholanthrene (1 nM) treatment to hepa 1 cells trahsfected with trout CYP1A-CAT construct stimulated the level of cyp1a1 mRNA by two folds and the activity of ethoxyresorufin-O-deethylase by two fold compared to that of control cells. In this study we reported that trout CYP1A-CAT reporter gene expression construct could be expressed by 3-methylcholanthrene treatment in mouse hepa 1 cells. Thus trout CYP1A-CAT could serve as a good model to study the mechanism of regulation of CYP1A1 gene expression.

• JSTS:Journal of Semiconductor Technology and Science
• /
• v.4 no.4
• /
• pp.251-257
• /
• 2004

Nonvolatile FRAMs with a design rule of 0.18 ${\mu}m$ were developed for the high performance smart card. A 1Mb FRAM was embedded in place of an EEPROM and a 64Kb FRAM was embedded in place of a. SRAM. It was confirmed that the FRAMs performed the roles of the EEPROM and SRAM successfully using the asynchronous write/read operation method and the one time programming (OTP) scheme. The cycle time of the FRAM was 10 MHz, which remarkably improved the write performance of the smart card in comparison with that of the conventional smart card with an EEPROM. Additionally, a simple and smart bit-line reference scheme for the future FRAM device having a 1T1C cell type was proposed.

• Journal of Ginseng Research
• /
• v.22 no.4
• /
• pp.310-315
• /
• 1998

The effect of red ginseng saponins (total saponins, Rbl- and Rgl- fraction of saponins) on the induction of $\beta$-galactosidase in yeast, hccharomyces cereuisiae, was investigated to see that ginseng saponins would penetrate the cell membrane and have a function in a nucleus as steroid hormones do. To attain such a kind of purpose, a DNA fragment (685bp) containing GALI promoter was inserted into the sites of EcoRl and BamHl of polylinker region, upstream of lace gene of the plasmid YEp356 (7.966 Kb), and thus the resulting plasmid pGALl-lacZ is supposed to express $\beta$- galactosidase only in the presence of galactose. The plasmid pGALl -lacZ was introduced into yeast, Ky106 (a leu2 ura3 his3 trp 1 Iys2), and the growth of the transformed cells was much slower in the presence of galactose than glucose. The effects of saponins on the specific activity of P-galactosidase from transformed yeast cells were detected. No significant increase was observed in case of total saponins, but the Rbl- or Rgl- fraction of saponins gave much higher increase in the activity. Maximum increase was observed as 35% in 10-3% of Rbl and as 75% in 10-1% of Rgl. These data suggest that ginseng saponins might be able to enter the nucleus and stimulate transcription. However, further studies to find out the putative saponin receptor are needed to confirm this possibility. Key words : Red ginseng saponin, $\beta$-galactosidase induction, Saccharomyces cerevisiae.

• Applied Microscopy
• /
• v.20 no.2
• /
• pp.57-70
• /
• 1990

Characterization and electron microscopic visualization of the plasmid and the gene expression of Escherichia coli were carried out. Transcriptional units of active structural genes were observed after lysis of Escherichia coli cells. The ribosomes attached to the E. coli genome on mRNA molecule as polyribosomes. From this gradient of polyribosome length, we estimated location of mRNA synthesis initiation site. In this experiment, a granule is ofen present which may correspond to a RNA polymerase at the promoter site. pOX1, pOX7, pOX7A, $pOX7{\Delta}1$, pSTP36, pSTP21, pBR322, and pJH12 were visualized by way of electron microscope, and their estimated sizes were determined to be $5.70{\pm}0.08{\mu}m,\;2.15{\pm}0.10{\mu}m,\;2.14{\pm}0.12{\mu}m,\;7.39{\pm}0.08{\mu}m,\;4.03{\pm}0.04{\mu}m,\;1.50{\pm}0.03{\mu}m\;and\;1.25{\pm}0.09{\mu}m$ respectively. One micrometer of measured length corresponded to about 3.0 Kb. Mica-press adsorption method that allows selectivs visualization of the plasmid DNA released in situ from the bacterial cell is rapid and useful for visualization of plasmids. The released plasmid DNA was adsorbed preferently on mica in a divalent cation-free solution. Miller chromatin-spreading method was useful to observe the plasmid and transcripts. BAC method and cytochrome C monolayer were useful to observe the plasmid DNA. Our ability to visualize ultrastructural aspects of the expression of E. coli has given us a unique tool with which to study the regulation the level of an individual gene.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.1
• /
• pp.153-159
• /
• 2001

In order to clone the peptide synthetase gene form Lysobacter lactamgenus IFO 14,288, the gene fragments were amplified using primers for the adenylation domain and the thionylation domain of the peptide synthetase genes in other organisms by polymerase chain reaction (PCR). The resulting 0.5-kb fragment was cloned in a pGEM-T vector, and the nucleotide sequences were determined. Six different PCR products were obtained; three were identified to be a part of L-$\alpha$-aminoadipyl-L-cysteinyl-D-valine (ACV) synthetase and three to be other peptide synthetases. Using each of the two different classes of PCR products as mixed probes, a cosmid library of L. lactamgenus chromosomal DNA constructed in a pHC79 vector was screened by an in situ hybridization procedure, and one positive clone was selected which was bound by peptide synthetase gene fragments as well as ACV synthetase gene fragments. The partial sequence analysis formt he obtained pPTS-5 cosmid showed th presence of more than two open reading frames. These were for two putative membrane transporters, which were homologous with several integral membrane proteins including the ABC transporter ATP-binding protein of E. coli (YbjZ) and the metal ion uptake protein of Bacillus subtilis (YvrN). A 45% homology was also found between the two transporter proteins at the carboxy terminus. Through a hydropathy analysis and transmembrane analysis. 4-5 transmembrane domains were found in these two proteins. When the genes were expressed in Escherichia coli, the gene products inhibited the hose cell growth, probably due to the disturbance of the membrane transport system.

• Korean Journal of Pharmacognosy
• /
• v.35 no.4 s.139
• /
• pp.288-292
• /
• 2004

To evaluate the quality of the crude drugs using three kinds of Cinnamomum Cortex (CC), Vietnamese CC (VCC, the stem bark of Cinnamomum obtusifolium), periderm-peeled Chinese CC (PPCC, periderm-peeled stem bark of C. cassia), Chinese CC (CCC, stem bark of C. cassia) and a Cinnamomi Ramulus (CR, the twig of C. cassia), the four essential oils were prepared by steam distillation method. Cinnamaldehdye (CAN) and an unknown substance tentatively named hydroxy-cinnamaldehdye(HCNA) were detected in the four essential oils by gas chromatography-mass spectrometry, the contents of which are significantly different one another. Vietnamese CC had the highest content of HCNA whereas CR had the highest CAN content and the lowest HCNA. Vietnamese CC exhibited the greatest cytotoxic activity against the cancer cell lines, A549, HepG-2, HL-60, P-388, U-937, and KB and CR the lowest cytotoxicity. Contents of CAN and HCNA in CCC and PPCC are positioned between VCC and CR. These results suggest that measurement of HCNA and cytotoxicity may determine the quality of CC and CR.

• Journal of Embryo Transfer
• /
• v.9 no.3
• /
• pp.243-247
• /
• 1994

최근 의약적으로 유용한 단백질을 대량 생산키 위한 실현 가능한 방법이 유전자변환 가축의 이용과 관련되어 발전되어 왔다. 이러한 유전자 변환동물은 이종의 단백질을 유즙속으로 분비시키는 생체반응기로서 이용되고 있다. 이러한 전략적 목적을 위해 현재 유전자 변환동물의 생산을 위한 이용에 있어 여러 가지 방법들이 보고되고 있다. 그러나 ES 세포의 사용이 이러한 방법들 사이에서 가장 실질적인 것으로 추정되고 있다. 본 실험에서는 유전자 구축을 위해 사람 황체 호르몬(human luteinizing hormone; hLH)의 전사를 유도하기 위해 각각 2.2 및 0.5 kb의 토끼 $\beta$-casein pronoter 단편을 이용하여 생쥐의 유선에 hLH를 발현시키도록 조절하고 발현이 thynidine kinase(TK) pronoter에 의해 좌우되는 neo 유전자를 selectable marker로서 plasnid속에 삽입하였다. 그 결과 생긴 구축 유전자는 각각 pCas 2.2와 pCas 0.5로 명명하였다. 구축된 유전자로 2$\times$107의 TT-2 ES세포를 170V, 550$\mu$F로 100$\mu$g의 선상 plasmid에 의해 electroporation 시켰다. 감염된 colony들은 250$\mu$g/$m\ell$ G418을 함유하는 ESM 배양액에서 선별 7일 이후에 회수하여 성공적으로 감염된 ES세포는 PCR 및 Southern blot에 의해 확인되었고 그들 중 나머지는 trypsin 처리 후 각각 미세조작과 공배양 기술을 사용하여 ICR 생쥐의 8세포기 수정란 속에 도입하였다. 결국 24시간 동안 37$^{\circ}C$, 5% $CO_2$에서 배양된 배반포를 chimera의 생산을 위해 위임신 유기된 G418 선발처리 이후 400 및 275개의 ES 세포 colony가 생존하였으며, 3개의 ES 세포으 colony 의 genome 속에 임의적으로 plamid가 삽입된 것을 Southern blot에 의해 확인되었다. 총 13 chimera 생쥐가 3 colony로부터 생산되었으나 germ-line chimera는 현재 조사중이다. chimera 생산빈도는 공배양 기술보다 주입방법에서 현저히 높았다.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.33 no.1
• /
• pp.1-9
• /
• 2011

Purpose: Tumor associated angiogenesis and/or lymphangiogenesis are known to be linked by VEGFR signaling pathways. These processes are regulated by several growth factors including VEGFR-2, VEGFR-3. E7080 is an orally active inhibitor of multiple tyrosine kinases including VEGFR-2, 3. Therefore, it was proposed that E7080 may inhibit angiogenesis and lymphangiogenesis. The aim of this study was to determine the effect of E7080 in a nude mouse model of OSCC. Methods: KB cells were xenografted into the submucosal tissue of the mouth floor of athymic mice. Seven days after the xenograft, the mice were randomized into 2 groups. E7080 were administered orally to the experimental group once per day. The mice were sacrificed 3 weeks after the treatment. The tumors were examined histopathologically. Immunohistochemical assays with anti- VEGF-C, VEGFR-2, VEGFR-3, phosphorylated VEGFR-2/3 (pVEGFR-2/3), and D2-40 antibodies were then performed. Results: The transplantation of human OSCC tumor cells into the mouth floor resulted in the formation of orthotopic tumors. The experimental (E7080 treatment) group showed a slowly increased tumor volume. Moreover, immunohistochemical staining demonstrated higher levels of VEGF-C, VEGFR-2, VEGFR-3, pVEGFR-2/3 and D2-40 expression in the control group than in the experimental group. Conclusion: These results suggest that E7080 may provide therapeutic benefits in OSCC.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.3
• /
• pp.827-830
• /
• 2013

Investigation of an organic extract of the fruits Schisandra wilsoniana led to the isolation of two new highly oxygenated nortriterpenoids, named schilancidilactones V-W (1-2). Their structures were elucidated by spectroscopic evidence. Compounds 1-2 feature a double bond between C-7 and C-8 compared with related known nortriterpenoids isolated from the genus Schisandra. Compounds 1 and 2 were tested for their anti-HIV-1 activities and cytotoxicity. The results revealed that compounds 1 and 2 showed moderate anti-HIV-1 activities with $EC_{50}$ 3.05 and 2.87 ${\mu}g/mL$, respectively, and compound 1 showed high cytotoxicity against KB and MDA-MB-231 cell with $IC_{50}$ values of 3.18 and 5.22 ${\mu}M$, respectively.