• 대한의용생체공학회:의공학회지
• /
• 제37권5호
• /
• pp.186-194
• /
• 2016

In this study, a co-axial flow induced microfluidic chip to fabricate pure collagen type I microfiber via the control of collagen type I and Na-alginate gelation process. The pure collagen type I microfiber was generated by selective degradation of Ca-alginate from 'Core-Shell' structured hydrogel microfiber. To make 'Core-Shell' structure, collagen type I solution was introduced into core channel and 1.5% Na-alginate solution was injected into side channel in microfluidic chip. To evaluatethe 'Core-Shell' structure, the red and green fluorescence substances were mixed into collagen type I and Na-alginate solution, respectively. The fluorescence substances were uniformly loaded into each fiber, and the different fluorescence images were dependent on their location. By immoblizing EpH4-Ras and C6 cells within collagen type I and Na-alginate solution, we sucessfully demonstrated the co-culture of EpH4-Ras and C6 cells with 'Core-Shell' like hydrogel microfiber for 5 days. Only to produce pure collagen type I hydrogel fiber, tri-sodium citrate solution was used to dissolve the shell-like Ca-alginate hydrogel fiber from 'Core-Shell' structured hydrogel microfiber, which is an excellent advantage when the fiber is employed in three-dimensional scaffold. This novel method could apply various application in tissue engineering and biomedical engineering.

• The Korean Journal of Physiology and Pharmacology
• /
• 제10권3호
• /
• pp.155-159
• /
• 2006

Among the Shc proteins, p66shc is known to be related to oxidative stress responses and regulation of the production of reactive oxygen species (ROS). The present study was undertaken to investigate the role of p66shc on endothelial nitric oxide synthase (eNOS) activity in the mouse embryonic fibroblasts (MEFs). When wild type (WT) or p66shc (-/-) MEFs were transfected with full length of eNOS cDNA, the expression and activity of eNOS protein were higher in the p66shc (-/-) MEFs. These phenomena were reversed by reconstitution of p66shc cDNA transfection in the p66shc (-/-) MEFs. The basal superoxide production in the p66shc (-/-) MEFs was not significantly different from that of WT of MEFs. However, superoxide production induced by NADPH in the p66shc (-/-) MEF was lesser than that in WT MEFs. When compared with WT MEFs, cell lysate of p66shc (-/-) MEFs showed significantly increased H-ras activity without change of endogenous H-ras expression. Our findings suggest the pivotal role of p66shc adaptor protein played in inhibition of endothelial nitric oxide production via modulation of the expression and/or activity of eNOS protein.

• BMB Reports
• /
• 제53권11호
• /
• pp.576-581
• /
• 2020

Dimethylation of the histone H3 protein at lysine residue 9 (H3K9) is mediated by euchromatin histone methyltransferase II (EHMT2) and results in transcriptional repression of target genes. Recently, chemical inhibition of EHMT2 was shown to induce various physiological outcomes, including endoplasmic reticulum stress-associated genes transcription in cancer cells. To identify genes that are transcriptionally repressed by EHMT2 during apoptosis, and cell stress responses, we screened genes that are upregulated by BIX-01294, a chemical inhibitor of EHMT2. RNA sequencing analyses revealed 77 genes that were upregulated by BIX-01294 in all four hepatic cell carcinoma (HCC) cell lines. These included genes that have been implicated in apoptosis, the unfolded protein response (UPR), and others. Among these genes, the one encoding the stress-response protein Ras-related GTPase C (RRAGC) was upregulated in all BIX-01294-treated HCC cell lines. We confirmed the regulatory roles of EHMT2 in RRAGC expression in HCC cell lines using proteomic analyses, chromatin immune precipitation (ChIP) assay, and small guide RNA-mediated loss-of-function experiments. Upregulation of RRAGC was limited by the reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC), suggesting that ROS are involved in EHMT2-mediated transcriptional regulation of stress-response genes in HCC cells. Finally, combined treatment of cells with BIX-01294 and 5-Aza-cytidine induced greater upregulation of RRAGC protein expression. These findings suggest that EHMT2 suppresses expression of the RRAGC gene in a ROS-dependent manner and imply that EHMT2 is a key regulator of stress-responsive gene expression in liver cancer cells.

• 한국수산과학회지
• /
• 제52권3호
• /
• pp.256-267
• /
• 2019

Styrofoam beads, which are relatively inexpensive and can provide a large specific surface area, were tested as filter media. Styrofoam beads with a diameter of $3{\pm}0.5mm$ were used; the specific surface area of the beads was $1,034m^2{\cdot}m^{-3}$. Five independent recirculating culture systems were used in the experiment. Each system consisted of one culture tank and three trickling bio-filters. Using the systems, nitrification efficiency was evaluated with respect to hydraulic loading rate (HLR) and carbon/nitrogen (C/N) ratio. The lowest ammonia and nitrogen concentrations were $0.84mg{\cdot}L^{-1}$ and $1.30mg{\cdot}L^{-1}$, respectively, observed at an HLR of $50.9m^3{\cdot}m^{-2}{\cdot}h^{-1}$. Nitrification efficiency in the culture tank was highest at a C/N ratio of 0, with ammonia and nitrite nitrogen concentrations of $0.32mg{\cdot}L^{-1}$ and $0.90mg{\cdot}L^{-1}$, respectively. Ammonia and nitrite nitrogen concentrations in the culture tank abruptly changed at C/N ratios ${\geq}3$.

• 한국독성학회:학술대회논문집
• /
• 한국독성학회 1998년도 국제심포지움 및 추계학술대회
• /
• pp.36-36
• /
• 1998

Tumorigenicity of benzo(a)pyrene (BP) and benzo(a)pyrene diol epoxides ((+)BPDE-1, (-)BPDE-1) was investigated in transgenic TG-AC mice carrying v-Ha-ras oncogene fused to the promoter of the mouse embryonic a-like, z-globin gene. Animals were topically treated twice per week for 25weeks with BPDE (10$\mu$g/mouse) and BP (10, 20, 40$\mu$g/mouse). In addition, animals were treated with BPDE or BP (initiated) followed by TPA (2$\times$2.5$\mu$g/week, for 4 weeks) for promotion study. In the continuous treatment of BPDE or BP, animals treated with 40$\mu$g BP showed $100\%$ tumor response after 20 weeks, $40\%$ of mice for 20$\mu$g BP, and $20\%$ for (+)BPDE-1, but (-)BPDE-1 and 10$\mu$g BP did not show any tumor response. After 25 weeks, most tumors turned out to be carcinomas in animals treated with 40$\mu$g BP. In BPDE or BP/TPA Initiation-promotion study, papilloma response occurred earlier (6 weeks after TPA treatment) than in continuously treated animals with BPDE or BP. RT-PCR assay for transgene expression showed that BP or BPOE was not transgene dependent in its tumorigenicity, but TPA was. Several Cytokine genes(TGF-a, TNF-a) and c-myc gene expressions were monitored in skin tissues during BP carcinogenesis. In early stage of BP treatment, the gene expressions were elevated(c-myc,TGF-a) or unchanged(TNF-a) compared to control, but the levels were gradually decreased during both middle and late stages of cacinogenesis, Gene expression levels of skin papillomas in acetone initiated-TPA promoted animals were close to those of middle stage or between middle and late stages. i-NOS was also highly expressed in carcinoma and papilloma, These data suggest that transgene expressions of TG-AC mice were not dependent on BP carcinogenesis and that TG-AC mice were more sensitive to TPA regardless of types of initiators. In addition, genes(TGF-a, c-myc, TNF-a, i-NOS) were modulated in the skin during BP cacinogenesis or TPA promotion.

• IMMUNE NETWORK
• /
• 제5권4호
• /
• pp.237-246
• /
• 2005

Background: Little information is available on the identification and characterization of the upstream regulators of the signal transduction cascades for Mycobacterium tuberculosis (M. tbc)-induced ERK 1/2 activation and chemokine expression. We investigated the signaling mechanisms involved in expression of CCL3 /MIP-1 and CCL4/MIP-1 in human primary monocytes infected with M. tbc. Methods: MAP kinase phosphorylation was determined using western blot analysis with specific primary antibodies (ERK 1/2, and phospho-ERK1/2), and the upstream signaling pathways were further investigated using specific inhibitors. Results: An avirulent strain, M. tbc H37Ra, induced greater and more sustained ERK 1/2 phosphorylation, and higher CCL3 and CCL4 production, than did M. tbc H37Rv. Specific inhibitors for mitogen-activated protein kinase (MAPK) kinase (MEK; U0126 and PD98059) significantly inhibited the expression of CCL3 and CCL4 in human monocytes. Mycobactetia-mediated expression of CCL3 and CCL4 was not inhibited by the Ras inhibitor manumycin A or the Raf-1 inhibitor GW 5074. On the other hand, phospholipase C (PLC) inhibitor (U73122) and protein kinase C (PKC)specific inhibitors ($G\ddot{o}6976$ and Ro31-8220) significantly reduced M. tbc-induced activation of ERK 1/2 and chemokine synthesis. Conclusion: These results are the first to demonstrate that the PLC-PKC-MEK-ERK, not the Ras-Raf-MEK-ERK, pathway is the major signaling pathway inducing M. tbc-mediated CCL3 and CCL4 expression in human primary monocytes.

• Journal of Microbiology and Biotechnology
• /
• 제14권6호
• /
• pp.1286-1294
• /
• 2004

In a previous study by the current authors, hepatocellular carcinoma (HCC) was determined to be epidemiologically sex-dependent, and the incidence and multiplicity of HCC found to decrease in estradiol-3 benzoate (EB)-treated F344 rats. Therefore, to ascertain the anticancer mechanism of EB, a commercially available cDNA microarray, with a total of 14,815 cDNA rat gene clones, was used to determine the differentially expressed genes in nontreated livers, EB-treated livers, and diethynitrosolamine (DEN)-induced HCC. In the sequenced experiment, a total of 85 genes were differentially expressed at either two or more times the rate of the normal expression, where 33 genes were downregulated by EB, and 52 genes upregulated. Candidate genes were selected according to significant changes observed in the mRNA expression in the EB-treated livers compared with the nontreated livers, then these genes were filtered according to their different expression patterns in the DEN-induced tumors compared to the estrogen-treated livers. To confirm the microarray data, a real-time PCR analysis was performed for ten selected genes: the H-ras revertant protein 107 (H­rev107), insulin-like growth factor binding protein (lOFBP), parathyroid hormone receptor (PI'HR), SH3 domain binding protein (SH3BP), metallothionein, src-suppressed C-kinase substrate (SSeCK) gene, phosphodiesterase I, CD44, epithelial membrane protein 3 (EMP3), and estrogen receptor a (ERa). The SSeCK and phosphodiesterase I genes were both upregulated in the DEN-induced hepatocarcinomas, yet their possible carcinogenic functions remain unknown. Meanwhile, the other genes were downregulated, including the genes related to growth regulation (IOFBP, H-revI07, ER$\alpha$), adipogenesis inhibition (PTHR), and tumor suppression (metallothionein).

• Toxicological Research
• /
• 제20권1호
• /
• pp.21-29
• /
• 2004

We used cDNA microarray to assess gene expression profiles in hematopoetic cell line, U-937, exposed to low doses of ionizing irradiation. The 1,000 DNA elements on this array were PCR-amplified cDNAs selected from named human cancer related genes. According to the strength of irradiation, the levels of some gene expression were increased or decreased as dose-dependent manner. The gene expressions of Tubulin alpha, protein kinase, interferon-alpha, -beta, -omega receptor and ras homolog gene family H were significantly increased. Especially, Tubulin gene was shown 2.5 fold up-regulated manner under stress of 500 rad irradiation than 200 rad. On the other hand, fibroblast growth factor 12 and four and a half LIM domains, etc. were significantly down-regu-lated. Also, tumor protein 53(TP53) related genes that p53 inducible protein, tumor protein 53-binding protein looks of little significance as radiation sensitive manner. The radio-sensitivity of tubulin gene etc. that we proposed could be useful to rapid and correct survey for the bio-damage by exposure to low dose irradiation.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
• /
• pp.180-180
• /
• 1994

Farnesyl protein transferase는 Ras precursor의 C-terminal에 있는 cystein residue에 farnesyl group을 결합시키는 효소다. 이 효소를 bovine testis에서 30-50% ammonium sulfate fractionation, DEAE-sephacel ion exchange, Sephacryl s-300 gel filtration, hexapeptide(KKCVIM) affinity chromatography를 통해 30000배로 분리하였다. 분리된 효소는 gel filtration시 약 100kDa으로, SDS-polyacrylamide 전기영동시 50kDa의 인접한 두 bands로 나타났고 이것은 $\alpha$, $\beta$ subunits으로 생각되었다. $\alpha$ subunit을 encoding하는 RAM2 유전자를 site directed mutagenesis로 145번의 histidine을 aspartate로, 140번의 aspartate를 asparagine 으로 바꾸었더니 optimal pH와 $K_{m}$ 값이 변했다. Diethyl pyrocarbonate로 histidine residues를 chemical modification시켰을때 효소의 활성이 저하되었다. 145번 histidine이 aspartate로 바뀐 돌연변이효소에서 비교적 느리게 활성이 저하되므로 145번 histidine이 이 효소의 active site에 있을것으로 추측된다.

• 한국식품과학회지
• /
• 제28권6호
• /
• pp.1038-1044
• /
• 1996

멸치액젓의 품질 및 위생적 안정성 유지, 그리고 장기저장 중의 저장성 향상을 위해 멸치액젓을 레토르트 식품화할 때 필요로 하는 가열처리의 최적 조건 및 레토르트 살균 멸치액젓 시제품의 성분조성, 그리고 저장 중 품질안정성 등에 대하여 실험하였다. 멸치액젓의 산도와 총질소량은 열처리의 정도가 커질수록 약간씩 감소하였고, 아미노질소량 역시 열처리 정도가 커질수록 감소한 반면, 휘발성 염기질소량은 열처리 정도가 커질수록 약간씩 증가하였다. 저장 1년동안 생멸치액젓의 휘발성 염기질소량은 약간 증가한 반면, 가열처리 시료들은 약간씩 감소하였다. pH는 생시료가 6.81, 가열처리 시료들은 $6.94{\sim}6.97$로서 가열처리 시료쪽의 pH가 약간 높았고, L, a, b 및 ${\Delta}E$값 측정 결과, 가열처리 시료들은 저장 1년동안 흑변과 같은 색조의 변화가 거의 일어나지 않았다. 생시료에서는 생균수가 $2.9{\sim}9.1{\times}10^3/ml$ 검출되었으나, Fo 3 이상의 가열처리 시료에서는 생균수가 검출되지 않았다. 제품의 유리아미노산으로 alanine, glutamic acid, leucine, isoleucine. valine, lysine 및 phenylalanine 등의 함량이 많았고, 가열처리의 정도가 커질수록 대부분의 유리아미노산이 일정함량씩 감소하는 경향을 보였다. IMP 및 hypoxanthin의 경우는 열처리에 따른 변화가 거의 없었으나, TMAO는 가열치리에 따라 열분해되어 감소하는 경향을 보인 반면, TMA는 약간씩 증가하였다. 가열처리에 따라 냄새성분은 저.중비점 휘발성 성분의 함량이 변화되거나 다른 냄새성분으로 변화되고 있음을 보여주었고, 또한 고비점 물질은 열처리의 정도가 커질수록 더욱 그 함량이 감소하는 경향을 나타내었다. 본 실험 결과, 멸치액젓을 레토르트 살균할 경우 Fo 3 이상의 열처리는 맛 및 냄새 면에서 상당한 관능적 품질저하를 초래하였으므로 적합하지 않았고, Fo 2 정도의 열처리가 저장 중 멸치액젓 특유의 풍미성분 및 위생적 안전성 유지, 그리고 품질안전성 향상 면에서 가장 유효한 것으로 판단되었다. 자숙처리 시료의 경우는 저장 중 냄새의 신선미가 약간 저하하는 것으로 나타났으나, 저장 1년 후에도 비교적 좋은 품질을 유지하는 것으로 평가되었다.