• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.136-136
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• 2003

Mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses, the overall success rate achieved by cloning techniques to date is low. This present study compared the incidences of DNA fragmentation during development of IVF, parthenotes (PT), nuclear transfer (NT) and transgenic (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counter staining was used for determination of DNA fragmentation and total number, respectively. TG and NT donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM+15% FCS until confluent, for 5 days. At 19 h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24 hpm with the combinations of ionomycin (5 M, 5 min) and cyclo-heximide (10 g/ml, 5 h) after electric fusion by a single DC pulse (1.6 KV/cm, 60 sec). Parthenotes were produced by the same activation protocol at 24 hpm. (중략)

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.4
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• pp.520-524
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• 1999

The present experiments were designed to examine whether exogenous DNA injected into the germinal crescent region (GCR) of early stage of developing embryos, which is considered to be the main place from which PGCs originate, can be transferred to recipient chicken embryos. In this experiment, Miw Z (DNA) dissolved in the transfection reagent (TR: Boehringer, Germany) was introduced into the GCR of donor embryos at stage 3-5 or 9-11, followed by continued incubation until the stage 13-15 of embryonic development. The PGCs collected from the embryonic blood vessels were examined for the incorporation of the injected DNA into the PGCs by the methods of X-gal staining and PCR analysis. As the results, the foreign DNA was successfully incorporated into the PGCS, leading to their transfer to the gonadal tissues. The present results, therefore, suggest that the early stage (3-5 or 9-11) of chicken embryonic development would be more successful than stage 13-15 in transferring exogenous genes to the recipient embryos, leading to the possibility of producing transgenic chicken medianting the PGCS.

• BMB Reports
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• v.32 no.6
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• pp.529-534
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• 1999

Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers desinged for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.

• Journal of Microbiology
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• v.38 no.1
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• pp.11-17
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• 2000

A new method was developed for the rapid analysis of diverse bacterial species in the natural environment. Our method is based on PCR-single-strands-conformation polymorphism (PCR-SSCP) and selective isolation technique of single-stranded DNA. Variable V3 fragments of 16S rDNA were amplified by PCR with bacterial 16S rDNA primers, where one of the primers was biotinylated at the 5'-end. The biotinylated strands of the PCR products were selectively isolated by using streptavidin paramagnetic particles and a magnetic stand, to prevent SSCP analysis producing heteroduplexes from heterogeneous DNA samples. The selected strands were separated by electrophoresis on a polyacrylamide gel, and detected by silver staining. Analysis of PCR products from 8 bacterial strains demonstrated their characteristic DNA band patterns. In addition, changes in the structure of the bacterial community and species diversity in the microcosm treated with phenol could be monitored. After 3 weeks of incubation, phenol and its intermediate, 2-hydroxy-muconic-semialdehyde, were degraded by indigenous bacteria. These dominating bacterial populations were identified as strong bands on an SSCP gel. Therefore, this study provides useful tools for microbial community analysis of natural habitats.

• Biomedical Science Letters
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• v.7 no.4
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• pp.181-190
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• 2001

This study was designed to investigate the effects of reactive oxygen species (ROS) on DNA stability in human spermatozoa. To verify human spermatozoa were incubated with xanthine-xanthine oxidase (X 100$\mu$M-XO 50 mlU ~ 400 mIU), $H_2O_2$ (125 $\mu$M ~ 1 mM), sodium nitroprusside (SNP 0.1 $\mu$M ~ 100 $\mu$M) or lymphocyte. Otherwise, spermatozoa were incubated under low $O_2$ (5%) condition. Damage of sperm DNA was analyzed by single cell electrophoresis (Comet assay) and flow cytometry after acridine orange staining. In the presence of ROS, there was increase in DNA damage. The rate of DNA single strand breakage (9.0$\pm$1.0% ~ 46.0$\pm$4.6%) and DNA fragmentation (7.51$\pm$1.0% ~ 29.5$\pm$4.6%) were similar regardless of the kinds of ROS and exposure time. DNA damage in the lower $O_2$ condition (5%) was lower than ambient $O_2$ condition (20%). Taken together, it suggested that sperm DNA might be damaged by ROS. In the presence of ROS, increase in DNA damage and chromatin instability was obvious in spite of short exposure. Although present study reconfirmed that sperm incubation in the low concentration of ROS have the benefit m the induction of capacitation and Ah, the increase in DNA damage by ROS and possible genetic problem should be considered before the human trials.

• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.6
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• pp.1025-1029
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• 2000

This study was conducted to investigate whether a DNA comet assay could be applied for identifying irradiated pork and beef. Pork and beef were irradiated with Co-60 gamma rays at 0.1, 0.3, 0.5, 0.7 and 1.0 kGy, and stored in a freezer Cells separated from the samples were embedded in agarose gel on a slide, dissolved in a lysis solution, and electrophoresed at 2 V/cm for 2.0 min by horizontal electrophoesis. The cells were then stained with a silver staining in order to visualize the DNA using a micro-scope. The DNA fragments of the irradiated cells stretched or migrated out of the cells and formed tails towards the anode, giving the appearance of comets, while unirradiated cells formed very short or no tails. The distance of DNA migration increased with irradiation dose. Since the statistical analysis showed a significant correlation between tail length and irradiation dose, a DNA comet assay could provide not only identification but also estimation of the irradiation dose for irradiated beef and pork.

• BMB Reports
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• v.34 no.3
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• pp.250-258
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• 2001

For a long time Rhus verniciflua Stokes (RVS) has traditionally been used as a herbal plant. It is known to contain various biological activities. Previously, a crude ethanol extract from RVS was reported to have antioxidant effects, and antiproliferative activities, on human cancer cell lines. In this report, we prepared a highly purified ethanol extract from RVS, which did not contain the urushiol derivatives, named REEE-1 ($\underline{R}$hus $\underline{e}$thanol $\underline{e}$luted $\underline{e}$xtract-1), to investigate the mechanisms of the scavenging activity of hydroxyl radicals using mouse thymocytes. The results from the deoxyribose, DNA nicking, and glucose/glucose oxidase enzyme assays showed that REEE-1 contained a strong scavenging activity of oxygen free radicals, especially of hydroxyl radicals. However, interestingly, REEE-1 also showed cytotoxicity against the thymocytes, although the effect was variable, depending on the concentrations and times of treatment. The REEE-1-mediated cytotoxicity against thymocytes, which has been used as one of the well-characterized models for apoptosis studies, was verified to be apoptotic. This was proven by the following: the appearance of DNA laddering, increases in DNA fragmentation, low fluorescence intensity in the nuclei after propidium iodide staining, and positive Annexin V staining of the cells. These results suggested that REEE-1 had both antioxidative activity and cytotoxicity against the thymocytes, although the effect of the cytotoxicity was variable, depending on the dose and time of the treatment.

• International Journal of Oral Biology
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• v.35 no.2
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• pp.51-60
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• 2010

Few studies have evaluated the apoptosis-inducing efficacy of NaF on cancer cells in vitro but there has been no previous investigation of the apoptotic effects of NaF on human oral squamous cell carcinoma cells. In this study, we have investigated the mechanisms underlying the apoptotic response to NaF treatment in the YD9 human squamous cell carcinoma cell line. The viability of YD9 cells and their growth inhibition were assessed by MTT and clonogenic assays, respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to detect apoptosis. YD9 cells were treated with NaF, and western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, and MMP and proteasome activity assays were performed sequentially. The NaF treatment resulted in a time- and dose-dependent decrease in YD9 cell viability, a dose-dependent inhibition of cell growth, and the induction of apoptotic cell death. The apoptotic response of these cells was manifested by nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, a decreased DNA content, the release of cytochrome c into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, a significant shift of the Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF treatment resulted in the downregulation of G1 cell cyclerelated proteins, and upregulation of p53 and the Cdk inhibitor $p27^{KIP1}$. Taken collectively, our present findings demonstrate that NaF strongly inhibits YD9 cell proliferation by modulating the expression of G1 cell cycle-related proteins and inducing apoptosis via mitochondrial and caspase pathways.

• Natural Product Sciences
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• v.10 no.1
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• pp.48-53
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• 2004

Two galloyl monosaccharides, 1,2,6-trigalloylglucose (1, TRgG) and 1,2,3,6- tetragalloylglucose (2, TEgG), were isolated from the stem-bark of Juglans mandshurica. Two galloylglucoses showed cytotoxic effects on human promyelocytic leukemia HL-60 cells. In order to elucidate their mechanism of action, we have investigated the flow cytometric analysis after Annexin V-FITC and PI staining, caspase-3 activity, and internucleosomal DNA fragmentation in HL-60 cells. HL-60 cells treated with both compounds 1 and 2 at 150 and $100\;{\mu}M$, respectively, led to a morphological features of apoptosis, such as plasma membrane blebbing and cell shrinkage. TRgG (1) and TEgG (2) increased the percentage of $FITC^+\;and\;FITC^+PI^+$ cells in flow cytometry after Annexin V-FITC and PI staining. The increase of apoptotic cells was preceded by the activation of caspase-3 reported to play a central role in apoptotic process and inducing internucleosomal DNA fragmentation. TEgG (2) showed to have stronger apoptosis inducing activity in HL-60 cell lines as compared with TRgG (1).

• Journal of Life Science
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• v.24 no.8
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• pp.903-909
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• 2014

Plants are an invaluable source of potential new anti-cancer drugs. Sasa quelpaertensis Nakai (Korean name, Jeju-Joritdae) is one of these plants with medical value, which is a bamboo grass widely distributed in Mt. Halla on Jeju Island, Korea. Here, we investigated the apoptotic effects of S. quelpaertensis leaf extracts in six human cancer cell lines (A549, MCF-7, HepG-2, Hela, HCT116 and A375). MTT assay signified the antiproliferative nature of S. quelpaertensis extracts against all tested cancer cells: S. quelpaertensis displayed slight cytotoxicity against A549, MCF-7 and HepG-2 cells, whereas it was exclusively cytotoxic to Hela, HCT116 and A375 cells. Apoptotic cells were evaluated using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). PI staining indicated increasing accumulation of Hela, HCT116 and A375 cells at sub-G1 phase. Further events like generation of nitric oxide ($NO^{\bullet}$) were accompanied in the S. quelpaertensis Nakai-induced apoptosis. Augmented $NO^{\bullet}$ generation resulted in the DNA fragmentation of Hela, HCT116 and A375 cells by treatment with S. quelpaertensis leaf extracts. These results suggest that S. quelpaertensis may be a potential natural resource for treating cancer cell. To identify the exact mechanisms of molecular mechanism of S. quelpaertensis induced apoptosis awaits further investigation.