• Animal Bioscience
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• v.36 no.7
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• pp.1022-1033
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• 2023

Objective: p66Shc, a 66 kDa protein isoform encoded by the proto-oncogene SHC, is an essential intracellular redox homeostasis regulatory enzyme that is involved in the regulation of cellular oxidative stress, apoptosis induction and the occurrence of multiple age-related diseases. This study investigated the expression profile and functional characteristics of p66Shc during preimplantation embryo development in sheep. Methods: The expression pattern of p66Shc during preimplantation embryo development in sheep at the mRNA and protein levels were studied by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The effect of p66Shc knockdown on the developmental potential were evaluated by cleavage rate, morula rate and blastocyst rate. The effect of p66Shc deficiency on reactive oxygen species (ROS) production, DNA oxidative damage and the expression of antioxidant enzymes (e.g., catalase and manganese superoxide dismutase [MnSOD]) were also investigated by immunofluorescence staining. Results: Our results showed that p66Shc mRNA and protein were expressed in all stages of sheep early embryos and that p66Shc mRNA was significantly downregulated in the 4-to 8-cell stage (p<0.05) and significantly upregulated in the morula and blastocyst stages after embryonic genome activation (EGA) (p<0.05). Immunofluorescence staining showed that the p66Shc protein was mainly located in the peripheral region of the blastomere cytoplasm at different stages of preimplantation embryonic development. Notably, serine (Ser36)-phosphorylated p66Shc localized only in the cytoplasm during the 2- to 8-cell stage prior to EGA, while phosphorylated (Ser36) p66Shc localized not only in the cytoplasm but also predominantly in the nucleus after EGA. RNAi-mediated silencing of p66Shc via microinjection of p66Shc siRNA into sheep zygotes resulted in significant decreases in p66Shc mRNA and protein levels (p<0.05). Knockdown of p66Shc resulted in significant declines in the levels of intracellular ROS (p<0.05) and the DNA damage marker 8-hydroxy2'-deoxyguanosine (p<0.05), markedly increased MnSOD levels (p<0.05) and resulted in a tendency to develop to the morula stage. Conclusion: These results indicate that p66Shc is involved in the metabolic regulation of ROS production and DNA oxidative damage during sheep early embryonic development.

• Radiation Oncology Journal
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• v.20 no.4
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• pp.367-374
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• 2002

Purpose : To investigate the growth inhibitory effects, and the underlying mechanism of human colon cancer cell (HT-29) death, induced by a new synthetic bile acid derivative (HS-1200). Materials and Methods : Human colon cancer cells (HT-29), in exponential growth phase, were treated with various concentrations of a new synthetic bile acid derivative (HS-1200). The growth inhibitory effects on HT-29 cells were examined using a frypan blue exclusion assay. The extent of apoptosis was determined using agarose gel electrophoresis, TUNEL assays and Hoechst staining. The apoptotic cell death was also confirmed by Western blotting of PARP, caspase-3 and DNA fragmentation factor (DFF) analysis. To investigate the involvement of mitochondria, we employed immunofluorescent staining of cytochrome c and mitochondrial membrane potential analyses. Results : The dose required for the half maximal inhibition $(IC_{50})$ of the HT-29 cell growth was $100\~150\;{\mu}M$ of HS-1200. Several changes, associated with the apoptosis of the HT-29 cells, were reveal by the agarose gel eletrophoresis, TUNEL assays and Hoechst staining, following their treatment with $100\;{\mu}M$ of HS-1200. HS-1200 treatment also induced caspase-3, PARP and DFF degradations, and the western blotting showed the processed caspase-3 p20, PARP p85 and DFF p30 and p11 cleaved products. Mitochondrial events were also demonstrated. The cytochrome c staining indicated that cytochrome c had been released from the mitochondria in the HS-1200 treated cells. The mitochondrial membrane potential $(\Delta\Psi_m)$ was also prominently decreased in the HS-1200 treated cells. Conclusion : These findings suggest that the HS-1200 - induced apoptosis of human colon cancer cells (HT-29) is mediated via caspase and mitochondrial pathways.

• Korean Journal of Animal Reproduction
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• v.27 no.4
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• pp.309-315
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• 2003

DNA methylation at CpG sites, which is a epigenetic modification, is associated with gene expression without change of DNA sequences. During early mouse embryogenesis, dynamic changes of DNA methylation occur. In this study, DNA methylation patterns of porcine embryos produced in vivo and in vitro were examined at various developmental stages by the immunocytochemical staining method. Interestingly, active demethylation was not observed on the paternal pronucleus of porcine zygotes. However, differences were detected in the passive demethylation process between in vivo and in vitro embryos. There was no change in the DNA methylation state until the blastocyst stage of in vivo embryos, whereas partial demethylation was observed in several blastomeres from a 4 cell stage to a morula stage of in vitro embryos. The whole genome of inner cell mass (ICM) and trophectoderm (TE) cells in porcine blastocysts were evenly methylated without de novo methylation. Our findings demonstrate that genome-wide demethylation does not occur in pig embryos during preimplantation development unlike murine and bovine embryos. It indicates that the machinery regulating epigenetic reprogramming may be different between species.

• The Journal of Korean Society for Radiation Therapy
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• v.4 no.1
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• pp.71-77
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• 1990

The present study was undertaken to investigate effects of ionizing radiation on DNA synthesis and chromosomal abnormality in cultured submandibular gland(SG) cells. SG cells from C57BL/6N Crj mice were cultured in Dulbecco's modified Eagle's medium (DME) supplemented with $10\%$ fetal bovine serum, antibiotics and fungizone. The cultured SG cells were irradiated with graded doses of gamma ray ($^{60}Co$) at a dose rate of 58.4rad/min. The effect of irradiation of $^{60}Co$ on DNA synthesis in cultured cells was evaluated by measuring the incorporation of 3H-TdR. Using conventional chromosome techniques and Giemsa staining methods, chromosomal abnormalities in cultured SG cells, induced by irradiation of $^{60}Co$ werw examined. Cytological observations were carried out by a light microscope with high resolving power. The results obtained were as follows : 1. DNA synthesis of SG cells was quantitatively dependent on a radiation dose compare to control. 2. A polyploids and few chromosome-type break, such as single and double breaks, deltions and triradial figures were more predominantly in irradiated SG cells than in control. This increase of chromosomal abnormality was in the proposition to the irradiation doses.

• 보존과학연구
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• s.20
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• pp.5-20
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• 1999

We have analyzed the allele and genotype frequencies from 10 fractions of ancient human skeleton in 3 pieces of Jar coffin excavated from Naju Bokamni3rd tumulus by PCR amplification, high resolution polyacrylamide gelelectorphoresis and silver staining. We could isolate human genomic DNA from 3 bone fractions but the rest of them could not be used as materials due to being decayed. We could detect sex determination as male and 3 genotypes of STR system, HUMTHO1, HUMTPOX and HUMC5F1PO from the bone fraction of left side in Jar coffin 3 and see the slightly reaction suggesting the sex as male from the bone fraction of the left side in Jar coffin 2 and female from the right side in Jar coffin 3.We have also analyzed the genotype frequencies of mitochondria from the bone fractions of the left side and the right side in jar coffin 3, respectively. From the result of indetifiying at nucletide position between 16018 and 16378of the base of hyper variable region(HV1) in the control region, We can presume that the both bones have the same maternal inheritance.

• The Journal of Internal Korean Medicine
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• v.33 no.3
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• pp.272-283
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• 2012

Objectives : The purpose of this study was to investigate the mechanism of protective effect of Jinmu-tang (JMT, Zhenwu-tang) extract on $H_2O_2$-induced cell death in C6 glial cells. Methods : Cultured C6 glial cells of white mice were pretreated with JMT extract and exposed to $H_2O_2$ for inducing cell death. We measure the cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and investigate the cell morphology using a light microscope after crystal violet (CV) staining. Reactive oxygen species (ROS) formation was analyzed using a flow cytometer and a fluorescent microscope after staining with 2'7'-dichlorofluorescein diacetate (DCF-DA). DNA fragmentation was analyzed using a flow cytometer after propidium iodide (PI) staining and nuclei morphology was investigated using a fluorescent microscope after 2-[4-amidinophenyl]-6-indo-lecarbamidine dihydrochloride (DAPI) staining. We analyzed expression of Bax, processing of procaspase-3 and poly (ADP-ribose) polymerase (PARP), and activation of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) by western blot method. Tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) secretion was analyzed using Quantikine kit. Results : We determined the elevated cell viability by JMT extract on $H_2O_2$-induced C6 glial cell death. ROS formation, DNA fragmentation, $I{\kappa}B{\alpha}$ phosphorylation, NF-${\kappa}B$ activation, and secretion of TNF-${\alpha}$ induced by $H_2O_2$ are inhibited by JMT extract pre-treatment. JMT extract inhibits Bax expression, processing of caspase-3 and PARP that are critical biochemical markers of apoptotic cell death. Conclusions : These results suggest that JMT extract has a protective effect on $H_2O_2$-induced C6 glial cell death in various pathways.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4135-4141
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• 2014

Background: Human papillomaviruses (HPV) may play an important role as one of the possible etiologies of oral squamous cell carcinoma (OSCC). The present study aimed to investigate the association between HPV and OSCC in young Japanese patients by examining the presence of HPV DNA and surrogate markers in OSCC tissues. Materials and Methods: Forty young patients with OSCC whose surgical specimens were available were analyzed and compared with 40 patients randomly recruited from a pool of patients aged >40 years. HPV DNA was detected using the polymerase chain reaction-based AMPLICOR$^{(R)}$ HPV test, and surrogate markers of HPV infection were analyzed using immunohistochemical techniques to detect $p16^{INK4a}$ and p53. Results: Only two (5%) young patients and one (2.5%) older patient were positive for HPV DNA. $p16^{INK4a}$ overexpression was identified in six (15%) young patients. p53 staining levels were not high in tissues of most young patients (27 patients, 67.5%). HPV DNA status did not significantly correlate with $p16^{INK4a}$ expression levels. Profiles of increased levels of $p16^{INK4a}$ expression with diminished levels of p53 staining were not associated with the presence of HPV DNA. The combined p53 with $p16^{INK4a}$ profiles were significantly correlated with alcohol consumption in younger patients (p=0.006). Conclusions: Results of the present study indicate that HPV is less likely to cause OSCC in young Japanese patients, and the $p16^{INK4a}$ expression level is not an appropriate surrogate marker for HPV infection in OSCC.

• Fisheries and Aquatic Sciences
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• v.20 no.11
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• pp.29.1-29.7
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• 2017

The objective of this study was to determine a simple and reliable ploidy identification protocol for the rainbow trout (RT), Oncorhynchus mykiss, in the field condition. To evaluate the ploidy level and compare different detection protocols, triploid RT and gynogenesis were induced by UV irradiation and/or heat shock. The hatching rate at day 30 was 85.2% and the survival rate at day 90 was 69.4% (fingerling). The sex ratio of female RT was 93.75% in the gynogenesis group, illustrating that the UV irradiation inactivated the sperm DNA. The hatching rate and survival rate were 82.0 and 74.7%, respectively, in the triploid-induced group. The triploid induction rate by heat shock procedure was 73.9%. Cytogenetic protocols for ploidy identification such as chromosome counting, erythrocyte nuclear size comparison, and analysis of nucleolar organizing regions (NORs) by silver staining were compared. Silver nitrate staining showed the greatest success rate (22/23 and 32/32 for the triploid-induced group and gynogenesis group, respectively), followed by erythrocyte nuclear size comparison (16/23 and 19/32 for the triploid-induced group and gynogenesis group, respectively) and, lastly, chromosome preparation (2/23 and 6/32 for the triploid-induced group and gynogenesis group, respectively) with the lowest success rate. Based on our findings, silver staining for RT ploidy identification is speculated to be highly applicable in a wide range of research conditions, due to its cost-effectiveness and simplicity compared to other numerous ploidy detection protocols.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.318.1-318.1
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• 2002

We found that 23-hydroxyursolic acid, triterpenoid was isolated from Cussonia bancoensis have a significant cytotoxic activity against HL -60 human promyelocytic leukemia cells. The IC of 23-hydroxyursolic acid was 32.83 $\mu$M. These anti-proliferative activity was due to induction of apoptosis. The effect of apoptosis was identified by DNA laddering, DAPI assay. PI staining, and Annerxin V-FITC binding assay. (omitted)

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.315-319
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• 2003

A novel method is developed to yield virus titers in 10 h, is easy to .perform using 96-well plates, and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immune-staining. The titer is determined by counting foci produced due to infection of virus under a fluorescent microscopy. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post infection time of 4 h. Therefore, 10 hours were enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer.