• Journal of Ginseng Research
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• v.43 no.1
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• pp.86-94
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• 2019

Background: Ginseng is believed to have antitumor activity. Autophagy is largely a prosurvival cellular process that is activated in response to cellular stressors, including cytotoxic chemotherapy; therefore, agents that inhibit autophagy can be used as chemosensitizers in cancer treatment. We examined the ability of Korean Red Ginseng extract (RGE) to prevent autophagic flux and to make hepatocellular carcinoma (HCC) cells become more sensitive to doxorubicin. Methods: The cytotoxic effects of total RGE or its saponin fraction (RGS) on HCC cells were examined by the lactate dehydrogenase assay in a dose- or time-dependent manner. The effect of RGE or RGS on autophagy was measured by analyzing microtubule-associated protein 1A/1B-light chain (LC)3-II expression and LC3 puncta formation in HCC cells. Late-stage autophagy suppression was tested using tandem-labeled green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3. Results: RGE markedly increased the amount of LC3-II, but green and red puncta in tandem-labeled GFP-mRFP-LC3 remained colocalized over time, indicating that RGE inhibited autophagy at a late stage. Suppression of autophagy through knockdown of key ATG genes increased doxorubicin-induced cell death, suggesting that autophagy induced by doxorubicin has a protective function in HCC. Finally, RGE and RGS markedly sensitized HCC cells, (but not normal liver cells), to doxorubicin-induced cell death. Conclusion: Our data suggest that inhibition of late-stage autophagic flux by RGE is important for its potentiation of doxorubicin-induced cancer cell death. Therapy combining RGE with doxorubicin could serve as an effective strategy in the treatment of HCC.

• Proceedings of the Korean Society of Life Science Conference
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• 2005.09a
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• pp.67-67
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• 2005

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.10a
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• pp.61-61
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• 2003

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1271-1278
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• 2012

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

• The Journal of Korean Society of Virology
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• v.28 no.3
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• pp.225-232
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• 1998

We have expressed GFP in Sf9 and Bm5 cells or Bombyx mori larvae by using Ac-Bm hybrid virus capable of replicating in both Bm5 and Sf9 cells. Genomic DNA of Ac-Bm hybrid virus expressing ${\beta}$-galactosidase was cotransfected with baculovirus transfer vector containing GFP gene, pBacPAK-GFP in Sf9 cells. The Ac-Bm hybrid virus harboring GFP was named as Ac-Bm hybrid virus-GFP. The Ac-Bm hybrid virus-GFP-infected insect cells were easily selected by detecting the emission of GFP from each well of cell culture dish on the UV illuminator. GFP produced by Ac-Bm hybrid virus-GFP in Sf9 and Bm5 cells or B. mori larvae was confirmed by SDS-PAGE and Western blot analysis using GFP antibody. In addition, B. mori larvae infected with Ac-Bm hybrid virus-GFP was apparently appeared fluorescence from the whole body at S days postinoculation. The fluorescence of GFP from the hemolymph and fat body of B. mori larvae infected with Ac-Bm hybrid virus-GFP was also observed by fluorescence microscope. In conclusion, our results demonstrated that in baculovirus expression vector system, use of Ac-Bm hybrid virus have an additional advantage of expanded host range for producing recombinant proteins.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.141-146
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• 2005

A new system for displaying proteins on the surface of Escherichia coli was developed using the Salmonella typhimurium outer membrane protein C (OmpC) as an anchoring motif. The C-terminal deletionfusion strategy was developed to fuse the polyhistidine peptides and green fluorescent protein (GFP) to the Cterminal of the truncated functional portion of OmpC. The polyhistidine peptides of up to 243 amino acids could besuccessfully displayed on the E. coli cell surface, which allowed recombinant E. coli to adsorb up to 34.2 μmol of Cd2+ per gram dry cell weight. The GFP could also be successfully displayed on the E. coli cell surface. These results suggest that the C-terminal deletion-fusion strategy employing the S. typhimurium OmpC as an anchoring motif provides a new efficient way for the display of large proteins on the surface of E. coli.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.283-292
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• 2009

Objective: Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study. Methods: Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested. Results: eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs. Conclusion: We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation characteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4263-4266
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• 2012

Colorectal cancer has become a major disease threatening human health. To establish animal models that exhibit the characteristics of human colorectal cancer will not only help to study the mechanisms underlying the genesis and development effectively, but also provide ideal carriers for the screening of medicines and examining their therapeutic effects. In this study, we established a stable, colon cancer nude mouse model highly expressing green fluorescent protein (GFP) for spontaneous metastasis after surgical orthotopic implantation (SOI). GFP-labeled colon cancer models for metastasis after SOI were successfully established in all of 15 nude mice and there were no surgery-related complications or deaths. In week 3, primary tumors expressing GFP were observed in all model animals under fluoroscopy and two metastatic tumors were monitored by fluorescent imaging at the same time. The tumor volumes progressively increased with time. Seven out of 15 tumor transplanted mice died and the major causes of death were intestinal obstruction and cachexia resulting from malignant tumor growth. Eight model animals survived at the end of the experiment, 6 of which had metastases (6 cases to mesenteric lymph nodes, 4 hepatic, 2 pancreatic and 1 mediastinal lymph node). Our results indicate that our GFP-labeled colon cancer orthotopic transplantation model is useful with a high success rate; the transplanted tumors exhibit similar biological properties to human colorectal cancer, and can be used for real-time, in vivo, non-invasive and dynamic observation and analysis of the growth and metastasis of tumor cells.

• Plant Biotechnology Reports
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• v.5 no.2
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• pp.157-167
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• 2011

In order to establish a reliable and highly efficient method for genetic transformation of pepper, a monitoring system featuring GFP (green fluorescent protein) as a report marker was applied to Agrobacteriummediated transformation. A callus-induced transformation (CIT) system was used to transform the GFP gene. GFP expression was observed in all tissues of $T_0$, $T_1$ and $T_2$ peppers, constituting the first instance in which the whole pepper plant has exhibited GFP fluorescence. A total of 38 T0 peppers were obtained from 4,200 explants. The transformation rate ranged from 0.47 to 1.83% depending on the genotype, which was higher than that obtained by CIT without the GFP monitoring system. This technique could enhance selection power by monitoring GFP expression at the early stage of callus in vitro. The detection of GFP expression in the callus led to successful identification of the shoot that contained the transgene. Thus, this technique saved lots of time and money for conducting the genetic transformation process of pepper. In addition, a co-transformation technique was applied to the target transgene, CaCS (encoding capsaicinoid synthetase of Capsicum) along with GFP. Paprika varieties were transformed by the CaCS::GFP construct, and GFP expression in callus tissues of paprika was monitored to select the right transformant.

• Journal of Korean Neurosurgical Society
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• v.29 no.6
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• pp.725-730
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• 2000

Purpose : The subcellular localization of E1B-19k has been known cytosol or nuclear membrane by immunohistochemical staining and could dimerize with Bax to regulate cell death also known by the in-vitro immunoprecipitation. We planed to confirm this dimerization of E1B-19k with Bax in vivo in Cos-7 cells by using green fluorescent protein. Material and Method : We cloned E1B-19k and Bax into C3-EGFP. C3-EGFP-E1B-19k, C3-EGFP-Bax, and C3-EGFP-E1B-19k and pcDNA3-Bax were transfected into Cos-7 cells. We explored location of E1B-19k and Bax, and confirmed its dimerization with Bax in transfected living healthy Cos-7 cells by following green fluorescent protein of E1B-19k on the confocal microscope. Results : E1B-19k was located diffusely in cytoplasm and in nucleus but not in mitochondria. It prevented cell death from the apoptosis by staurosporine but its location was not changed. GFP-E1B-19k is not changed its intracellular location with Bax even with staurosporine. Conclusion : These results support that E1B-19k does not localize in mitochondria nor dimerize with Bax even with staurosporine. We could anticipate E1B-19k prevent cell death via the other dimerizing partner or pathways.