• The Journal of the Korean Society for Microbiology
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• v.3 no.1
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• pp.51-65
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• 1968

The site of the synthesis of Japanese encephalitis virus(JEV) in the actinomycin-treated and infecter PS Y15 cells(a porcine kidney stable cell line) was observed by the immunofluorescent antibody technique, acridine orange staining, and the autoradiographic analysis. In the parallel studies by immunofluorescent technique and acridine orange staining it the infected cells, Viral protein(as an antigen) and viral RNA were detected at the same site of cytoplasm. In the autoradiographic analysis, the cytoplasmic labeling of $^3H$-uridine was due to the synthesis of JEV-RNA, while the nucleolus and nucleus were not involved. In the autoradiographic studies on the secton of infected cells, the $^3H$-uridine was frequently incorporated around the cytoplasmic vacuoles. This localization of labeling agreed with the site of acridine orange positive granules. The results suggest that the syntheses of the viral RNA and viral protein occurred in the similar site of cytoplasm of the infected cells, and also the virus particles seem to be assembled in the sites of the viral RNA and protein syntheses.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.6
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• pp.524-529
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• 2006

For the repairing of bone defect, autogenous or allogenic bone grafting remains the standard. However, these methods have numerous disadvantages including limited amount, donor site morbidity and spread of diseases. Tissue engineering technique by culturing stem cells may allow for a smart solution for this problem. Adipose tissue contains mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from buccal fat pad and differentiate them into osteoblast and are to examine the bone induction capacity. Buccal fat-derived cells (BFDC) were obtained from human buccal fat pad and cultured. BFDC were analyzed for presence of stem cell by immunofluorescent staining against CD-34, CD-105 and STRO-1. After BFDC were differentiated in osteogenic medium for three passages, their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase (ALP) staining, Alizarin red staining and RT-PCR for osteocalcin (OC) gene expression. Immunofluorescent and biochemical assays demonstrated that BFDC might be a distinguished stem cells and mineralization was accompanied by increased activity or expression of ALP and OC. And calcium phosphate deposition was also detected in their extracelluar matrix. The current study supports the presence of stem cells within the buccal fat pad and the potential implications for human bone tissue engineering for maxillofacial reconstruction.

• Journal of Ginseng Research
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• v.47 no.3
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• pp.448-457
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• 2023

Background: Alzheimer's disease (AD) is a common form of dementia, and impaired mitophagy is a hallmark of AD. Mitophagy is mitochondrial-specific autophagy. Ginsenosides from Ginseng involve in autophagy in cancer. Ginsenoside Rg1 (Rg1 hereafter), a single compound of Ginseng, has neuroprotective effects on AD. However, few studies have reported whether Rg1 can ameliorate AD pathology by regulating mitophagy. Methods: Human SH-SY5Y cell and a 5XFAD mouse model were used to investigate the effects of Rg1. Rg1 (1µM) was added to β-amyloid oligomer (AβO)-induced or APPswe-overexpressed cell models for 24 hours. 5XFAD mouse models were intraperitoneally injected with Rg1 (10 mg/kg/d) for 30 days. Expression levels of mitophagy-related markers were analyzed by western blot and immunofluorescent staining. Cognitive function was assessed by Morris water maze. Mitophagic events were observed using transmission electron microscopy, western blot, and immunofluorescent staining from mouse hippocampus. The activation of the PINK1/Parkin pathway was examined using an immunoprecipitation assay. Results: Rg1 could restore mitophagy and ameliorate memory deficits in the AD cellular and/or mouse model through the PINK1-Parkin pathway. Moreover, Rg1 might induce microglial phagocytosis to reduce β-amyloid (Aβ) deposits in the hippocampus of AD mice. Conclusion: Our studies demonstrate the neuroprotective mechanism of ginsenoside Rg1 in AD models. Rg1 induces PINK-Parkin mediated mitophagy and ameliorates memory deficits in 5XFAD mouse models.

• The Journal of the Korean Society for Microbiology
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• v.21 no.3
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• pp.399-406
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• 1986

This study was performed to establish human plasmacytoma cell line as the partner cells for producing human hybridoma. Bone marrow cells from a multiple myeloma patient from Seoul National University Hospital, Korea were cultured and established as the cell line, named as HMC-BM4. HMC-BM4 cells were cultivated in RPMI 1640 media containing 8-azaguanine(8-AG; gradually increasing concentration from $1\;{\mu}g/ml$ to $20\;{\mu}g/ml$). 8-AG resistant cells were collected and cloned by limiting dilution. Each clone was divided and tested to die in hypoxanthine, aminopterine and thymidine (HAT) selection media. Finally one clone was selected and named as HMC-AR, which was sensitive to HAT selection media. HMC-AR cells showed typical morphology of plamacytoma in Wright staining. No cell formed the rosette with sheep erythrocytes. Surface membrane $\mu$ heavy chain was detected in 20% of HMC-AR cells and cytoplasmic $\mu$ heavy chain in 90% of them by direct immunofluorescent staining. Ia-like antigen was found in 90% of HMC-AR cells by indirect immunofluorescent staining using anti-Ia-like antigen monoclonal antibody, 1BD9-2. And about $1.0\;{\mu}g/ml$ of human $\mu$ heavy chain was detected in the 3-day culture supernatant of HMC-AR cells. 88% of cells contained 46 chromosomes. Mycoplasma was not detected in HMC-AR cells by Hoechst 33258 staining. This cell line would be used for making hybridomas secreting human monoclonal antibody.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.4
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• pp.331-339
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• 2007

Background: Distraction osteogenesis(DO) is a useful method for treating cases demanding the generation of new bone. During DO, the angiogenic activity is crucial factor in the new bone formation. The aim of this study was to detect the autocrine growth activity in the cellular components of the distracted bone with observation of the co-expression of vascular endothelial growth factor(VEGF) and its receptors following the mandibular DO. Materials and methods: Unilateral mandibular distraction(0.5 mm twice per day for 10 days) was performed in six mongrel dogs. Two animals were killed at 7, 14, and 28 days after completion of distraction, respectively. Immediately after the animals were killed, the right mandibles were harvested en block. Immunohistochemical staining was processed for observation of the VEGF expression, and double immunofluorescent staining was also processed for detection of the co-expression of osteocalcin and VEGF's two distinct receptors(VEGFR-1 and VEGFR-2). Results: At 7 and 14 days after distraction, the expressions of VEGF were significantly increased in the osteogenic cells of the distracted bone. Up to 28 days after distraction, VEGF was still expressed moderate in the osteoblastic cells of distracted bone. The co-expressions of osteocalcin/VEGFR-1 and osteocalcin/VEGFR-2 were observed in the distracted bone at 7 and 14 days after distraction. In the double immunofluorescent staining, the co-expression' s level of osteocalcin/VEGFR-1 was more than that of osteocalcin/VEGFR-2. Conclusion: Taken together, this study suggested that VEGF plays an important role in the osteogenesis, and these osteoblastic cell-derived VEGF might act as autocrine growth factor during distraction osteogenesis. In the other word, the cellular components, such as osteoblasts and immature fibroblast-like cellsor mesenchymal cells in the distracted bone, might have autocrine growth activity during distraction osteogenesis.

• Journal of Acupuncture Research
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• v.27 no.4
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• pp.29-38
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• 2010

Objectives : The purpose of the study is to determine the neuroprotective effect of modified Boyanghwano-tang(mBHT), a traditional Korean medicine, on the transient focal cerebral ischemia in rats. Methods : Focal ischemia and reperfusion were induced by middle cerebral artery occlusion(MCAO) for 90 min, followed by 144 h reperfusion in rats. mBHT(200mg/kg body weight, p.o.) was administrated in rats once a day during reperfusion. At the end of treatment, brain infarction was measured by TTC staining, and histological change was observed by H&E staining. The expressions of Bax, Bcl-2 and cytochrome c in ischemic brains were determined by immunofluorescent analysis. Results : mBHT significantly reduced the cerebral infarct volumes of the MCAO rats. mBHT also attenuated the neuronal cell death and the expressions of pro-apoptotic molecules, bax and cytochrome c in ischemic brains. Further, mBHT significantly increased the survival time of ischemeic rats and the expression of anti-apoptotic molecule, Bcl-2 in ischemic brains. Conclusions : Our results suggest that mBHT is neuroprotective and may prove to be useful adjunct in the treatment of ischemic stroke.

• Archives of Plastic Surgery
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• v.37 no.1
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• pp.7-11
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• 2010

Purpose: Vascular endothelial growth factor (VEGF) is known as a growth factor of endothelium and fibroblast. The purpose is to know the VEGF effects on fibroblast proliferation and fibroblast's notch receptor expression. Methods: CCD-986sk fibroblast was purchased from the Korean Cell Bank and was used in XTT assay for proliferation and wound healing assay for migration. Immunofluorescent (IF) staining and western blotting were used in testing notch expression of fibroblast. Semiquantitative RT-PCR was used in checking notch 1 mRNA production by fibroblast. Student-t test was used for analyzing results. Results: Cell proliferation assay using XTT showed significant higher proliferation in VEGF treated fibroblast, $2.324{\pm}0.0026$ vs. $2.463{\pm}0.017$ (p=0.002). Wound healing assay showed longer migration in VEGF treated fibroblast (p=0.062). The fluorescence was brighter in VEGF treated cells of notch 1 IF staining. Notch 1 expressions and mRNA productions increased more in VEGF treated cells. Conclusion: VEGF stimulates fibroblast to proliferate, migrate and to express Notch 1 simultaneously. Notch receptor could be related to VEGF mediated wound healing.

• Clinical and Experimental Pediatrics
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• v.51 no.10
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• pp.1112-1117
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• 2008

Purpose : We intended to observe cell death and apoptotic changes in neurons in organotypic hippocampal slice cultures following oxygen-glucose deprivation (OGD), using propidium iodide (PI) uptake, Fluoro-Jade (FJ) staining, TUNEL staining and immunofluorescent staining for caspase-3. Methods : The hippocampus of 7-day-old rats was cut into $350{\mu}m$ slices. The slices were cultured for 10 d (date in vitro, DIV 10) and and exposed to OGD for 60 min at DIV 10. They were then incubated for reperfusion under normoxic conditions for an additional 48 h. Fluorescence of PI uptake was observed at predetermined intervals, and the cell death percentage was recorded. At 24 h following OGD, the slices were Cryo-cut into $15{\mu}m$ thicknesses, and Fluoro-Jade staining, TUNEL staining, and immunofluorescence staining for caspase-3 were performed. Results : 1) PI uptake was restricted to the pyramidal cell layer and DG in the slices after OGD. The fluorescent intensities of PI increased from 6 to 48 h during the reperfusion stage. The cell death percentage significantly increased time-dependently in CA1 and DG following OGD (P<0.05). 2) At 24 h after OGD, many FJ positive cells were detected in CA1 and DG. Some neurons had distinct nuclei and processes while others had fragmented nuclei and disrupted processes in CA1. TUNEL and immunofluorescent staining for caspase-3 showed increased expression of TUNEL labeling and caspase-3 in CA1 and DG at 24 h after OGD. Conclusion : The numerous dead cells in the slice cultures after OGD tended to display apoptotic changes mediated by the activation of caspase-3.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.628-634
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• 2002

A microfilament-based motility in Toxoplasma gondii (T. gondii) Is involved in host cell invasion, yet the exact mechanism has not yet been determined. Accordingly, the current study examined the localization of actin and tubulin in T gondii using immunofluorescent (IF) and immunogold staining for electron microscopy. Indirect immunofluorescence (IF) staining using anti-actin and anti-tubulin monoclonal antibodies (mAbs) revealed localization of fluorescence on the entire surface of the tachyzoites. The actin in T. gondii was observed by immunogold staining, and the gold particles were seen on the surface, especially at the anterior end and in the cytoplasm of the parasite. However, there were no gold particles in the nucleus, rhoptries, and dense granules. The tubulin in T gondii was located on the surface and in the cytoplasm of the tachyzoites in the extracellular parasite, compared with anterior part of tachyzoites in the intracellular parasite. The antigens of T gondii recognized by anti-actin mAb were 107 kDa, 50 kDa, 48 kDa, and 40 kDa proteins, while those recognized by anti-tubulin mAb were 56 kDa, 52 kDa, and 34 kDa proteins. Tachyzoites of T gondii pretreated with the actin inhibitor, cytochalasin D (20 $\mu\textrm{g}$/ml), and tubulin inhibitor, colchicine (2$\times$10$\^$-6/ M), for 30 min at 37$\^{C}$ were used to infect the isolated mouse macrophages (tachyzo ites:macrophage=2:1). Pretreatment with the inhibitors resulted in lower multiplication of tachyzoites within the macrophages than in the untreated group 18 h post infection (p<0.05). Therefore, the present results suggest that actin and tubulin appear to be involved in the invasion of and multiplication in host cells.

• 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.