• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.205-209
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• 2008

This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine $\beta$-casein locus. For construction of knock-in vector using porcine $\beta$-casein gene, we cloned the $\beta$-casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5' recombination arm and a 2.7-kb fragment as the 3' recombination arm. We used the neomycin resistance gene ($neo^{r}$) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.

• Molecular & Cellular Toxicology
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• v.2 no.3
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• pp.176-184
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• 2006

The controversy on genotoxicity of molinate, an herbicide, has been reported in bacterial system, and in vitro and in vivo mammalian systems. To clarify the genotoxicity of molinate, we performed bacterial gene mutation test, in vitro chromosome aberration and mouse lymphoma $tk^{+/-}$ gene assay, and in vivo micronucleus assay using bone marrow cells and peripheral reticulocytes of mice. In bacterial gene mutation assay, no mutagenicity of molinate ($12-185{\mu}g/plate$) was observed in Salmonella typhimurium TA 98, 100, 1535 and 1537 both in the absence and in the presence of S-9 metabolic activation system. The clastogenicity of molinate was observed in the presence ($102.1-408.2\;{\mu}g/mL$) of metabolic activation system in mammalian cell system using Chinese hamster lung fibroblast. However, no clastogenicity was observed in the absence ($13.6-54.3\;{\mu}g/mL$) of metabolic activation system. It is suggested that the genotoxicity of molinate was derived some metabolites by metabolic activation. Molinate was also subjected to mouse lymphoma L5178Y $tk^{+/-}$ cells using microtiter cloning technique. In the absence of S-9 mixture, mutation frequencies (MFs) were revealed $1.4-1.9{\times}10^{-4}$ with no statistical significance. However, MFs in the presence of metabolic activation system revealed $3.2-3.4{\times}10^{-4}$ with statistical significance (p<0.05). In vivo micronucleus (MN) assay using mouse bone marrow cells, molinate revealed genotoxic potential in the dose ranges of 100-398 mg/kg of molinate when administered orally. Molinate also subjected to acridine orange MN assay with mouse peripheral reticulocytes. The frequency of micronucleated reticulocytes (MNRETs) induced 48 hr after i.p. injection at a single dose of 91, 182 and 363 mg/kg of molinate was dose-dependently increased as $10.2{\pm}4.7,\;14.6{\pm}3.9\;and\;28.6{\pm}6.3\;(mean{\pm}SD\;of\;MNRETs/2,000\;reticulocytes)$ with statistical significance (p<0.05), respectively. Consequently, genotoxic potential of molinate was observed in in vitro mammalian mutagenicity systems only in the presence of metabolic activation system and in vivo MN assay using both bone marrow cells and peripheral reticulocytes in the dose ranges used in this experiment. These results suggest that metabolic activation plays a critical role to express the genotoxicity of molinate in in vitro and in vivo mammalian system.

• Journal of Animal Science and Technology
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• v.45 no.5
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• pp.695-702
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• 2003

Nucleolus Organizer Regions (NORs) are the specific chromosome sites where ribosomal genes are located and highly expressed. We have applied the AgNOR staining to identify the distribution of NORs in the chromosomes of Korean Cattle. We have also studied the NORs pattern on the cells originated from different breeds, tissues and sex. Peripheral blood from forty-four Korean Cattle and Holstein was cultured for chromosme preparation. The fibroblast culture from biopsied ear skins was also conducted for chromosome analysis. The distribution of NORs was analyzed by sequential Ag staining and G-banding on metaphases of the cells. In Korean Cattle, the NORs are localized on the telomeres of the five chromosome pairs number 2, 3, 4, 11 and 28. The number of NORs per metaphase ranged from 2 to 10 giving a mean value of 5.6. The number of NORs per cell varied among individuals and cells within same individual. The size of NORs also differed in NO-chromosomes. The number of NORs was significantly different between Korean Cattle and Holstein, fibroblasts and lymphocytes, and male and female. However, the distribution and frequency of NORs were similar among the cells regardless of breeds, tissues, and sex.

• Polymer(Korea)
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• v.36 no.5
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• pp.586-592
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• 2012

Iron oxides ($Fe_3O_4$) are metabolically secreted after endocytosed by cells, indicating no cytotoxicity. Therefore, they are widely used as a contrast agent before photographing of magnetic resonance imaging. In this study, iron oxide nanoparticles are synthesized by the co-precipitation method and subsequently immobilized with a homing peptide (AP), which specifically interacts with interleukin-4 receptor located on the membrane of endothelial and bladder cancer cells. The size of AP-immobilized iron oxide particle is about 39 nm. Intracellular uptake of the AP-immobilized iron oxide nanoparticles was investigated using bladder cancer cells and fibroblasts as the control. As the result, the nanoparticles are specificially uptaken by bladder cancer cells. However, the nanoparticles are not specificially uptaken by fibroblast. It could be said that the AP-immobilized iron oxide nanoparticles have a potential to be used as a contrast agent for early diagnosis of cancer.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.21 no.4
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• pp.332-344
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• 1999

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. Interleukin-6 (IL-6) is involved in the final differentiation of B cells into antibody-producing cells. Interleukin-8 (IL-8) is a neutrophil chemotactic factor that plays an important role in the recruitment of neutrophil to inflammatory loci. Inflammatory mediators by cells in the gingiva have been implicated in the initiation and progression of periodontitis and oral infection. The purpose of this study was conducted to investigate the effect of lipopolysaccharide (LPS), staphylococcus enterotoxin B (SEB) on production of IL-6 and IL-8 by human gingival and facial dermal fibroblasts. Primary cultured human gingival and facial dermal fibroblasts were incubated with LPS (0.01, 0.1, $1.0{\mu}g/ml$), SEB (0.01, 0.1, $1.0{\mu}g/ml$) or LPS $(0.1{\mu}g/ml)$ plus SEB $(0.1{\mu}g/ml)$. Culture supernatants were collected at 24, 48, and 72 hrs and assessed for IL-6 and IL-8 production by enzyme-linked immunosorbent assay. IL-6 production in gingival fibroblasts stimulated with LPS was higher than that with SEB. IL-6 production by double exposure with LPS plus SEB was amplified in comparison with single exposure of LPS or SEB. IL-6 production in facial dermal fibroblasts was increased only by stimulation with a high concentration of LPS $(1.0{\mu}g/ml)$. Its production in facial dermal fibroblasts by exposure with SEB was decreased in comparison with control, nontreated cells. Therefore, gingival fibroblasts showed higher sensitivity than facial dermal fibroblasts in response to low concentration of LPS. Also, IL-6 production by double exposure with LPS plus SEB was amplified in comparison with single exposure of LPS or SEB. IL-8 production in gingival fibroblasts was enhanced greatly only by stimulation of high concentration of LPS $(1.0{\mu}g/ml)$. That by exposure with SEB was increased only in 24 hrs cultivation. IL-8 production by double exposure with LPS plus SEB was amplified in comparison with single exposure of LPS or SEB. IL-8 production in facial dermal fibroblasts was decreased by LPS and increased only in 48 hrs cultivation by SEB. IL-8 production by double exposure with LPS plus SEB was enhanced only in 48 hrs cultivation in comparison with single exposure of LPS or SEB. therefore, IL-6 and IL-8 production were released at various quantities according to bacterial toxin applied and site of fibroblast harvested. These results suggest that gingival fibroblasts may be concerned with IL-6 and IL-8 related inflammatory response more than facial dermal fibroblasts.

• Journal of Periodontal and Implant Science
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• v.31 no.3
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• pp.597-610
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• 2001

Normal gingival fibroblasts functioning is fundamental for the maintenance of periodontal connective tissue as well as wound healing. Nicotine have been found to affect DNA synthesis and cell proliferation, which appear to depend on the type of cells. This in vitro study was done to determine the effects of nicotine, a major component of tobacco, on cell proliferation, viability, activity, cell cycle distribution, and expression of cell cycle regulatory proteins in human gingival fibroblasts. Nicotine has been tested for 2 days or 4 days in 5 different concentrations; $0.1{\mu}g/ml$; $1{\mu}g/ml$; $10{\mu}g/ml$; $100{\mu}g/ml$; $1000{\mu}g/ml$. To assess cell proliferation and viability, viable and non-viable cells were counted by hemocytometer; to evaluate cellular activity, MTT assay was employed; to analyze cell cycle distribution, fluorescent propidium iodide-DNA complex were measured using fluorocytometer; to determine the expression of cell cycle regulatory proteins, western blot analysis was performed. After 2 days and 4 days incubation respectively, at concentrations of $1{\mu}g/ml$ - $1000{\mu}g/ml$, nicotine significantly inhibited proliferation comparing to non-supplemented controls. The cell viability was significantly decreased after 2 days and 4 days at concentrations of $1{\mu}g/ml$ - $1000{\mu}g/ml$ and at $10{\mu}g/ml$ - $1000{\mu}g/ml$ respectively. After 2 days and 4 days, the cellular activity was significantly decreased at concentrations of $10{\mu}g/ml$ - $1000{\mu}g/ml$. Treatment with $100{\mu}g/ml$ nicotine for 48 hours caused an increase in the proportion of G1-phase cells (from 46.41% to 53.46%) and a decrease in the proportion of S-phase cells (from 17.80% to 14.27%). The levels of cyclin $D_1$ and CDK 4 proteins in nicotine-treated fibroblasts were lower than that of controls, whereas the levels of p16 and pRB were higher than that of controls. These results suggest that the decrease of cell proliferation and lengthened Gap phases (G1) by nicotine may due to the increased expression of p16 and pRB as well as decreased expression of cyclin $D_1$ and CDK 4 in human gingival fibroblasts.

• Archives of Pharmacal Research
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• v.21 no.4
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• pp.391-397
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• 1998

The toxicity evaluation of oriental herbal drugs is of great concern at present. Bojungchisup-tang (BCST, in Korean), a decocted medicine of oriental herbal mixture, is now well used in clinic at oriental hospitals for the treatment of edema of several diseases in practice. However, the toxicity of the oriental herbal decocted medicines such as genetic toxicity is not well defined until now. In this respect, to clarify the genetic toxicity of BCST, in vitro chromosome aberration assay with Chinese hamster lung (CHL) fibroblasts and in vivo supravital micronucleus assay with mouse peripheral reticulocytes were performed in this study. In the chromosome aberration assay, we used 5,000 $\mu\textrm{g}$/ml BCST as maximum concentration because no remarkable cytotoxicity in CHL cells was observed both in the presence and absence of S-9 metabolic activation system. No statistical significant differences of chromosome aberrations were observed in CHL cells treated with 5,000, 2,500 and 1,250 $\mu\textrm{g}$/ml BCST for 6 hour both in the presence and absence of S-9 metabolic activation. However, very weak positive result (6.5-8.0% aberration) of BCST was obtained in the absence of S-9 metabolic activation system at 5,000 $\mu\textrm{g}$/ml BCST when treated for 24 hour, i.e. 1.5 normal cell cycle time. And also, in vivo clastogenicity of BCST was studied by acridine orange-supravital staining micronucleus assay using mouse peripheral reticulocytes. We used 2,000 mg/kg as the highest oral dose in this micronucleus assay because no acute oral toxicity of BCST was observed in mice. The optimum induction time of micronucleated reticulocytes (MNRETS) was determined as 36 hours after oral administration of 2,000 mg/kg BCST. No significant differences of MNRETs between control and BCST treatment groups were observed in vivo micronucieus assay. From these results, BCST revealed very weak positive result in chromosome aberration assay in vitro with CHL cells and no clastogenicity in micronucieus assay in vivo.

• Korean Journal of Medicinal Crop Science
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• v.11 no.1
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• pp.31-39
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• 2003

The antioxidative activities and biological properties in the EtOAc extracts and purified extracts of Orostachys japonicus were measured by assay of DPPH, xanthine/xanthine oxidase and mammalian cells(2-12). Scavenging of DPPH radical and inhibition of xanthine/xanthine oxidase of EtOAc extracts were showed the highest activity in the arable land and in September. The S-4 fraction showed the highest activity among the silica-gel column chromatography fractions. LH-4 fraction showed higher activity than the other fractionsins in assay of DPPH and xanthine/xanthine oxidase. Fatty acids and phenolic compounds were identified by GC/MS and main compounds were 1,2,3-benzenetriol, alpha-androsta-7,14-diene in LH-4 fraction. The activities of POD and SOD in samples havested on different habitats were high such as arable land> intermountain> seashore. That of POD and SOD in crude extracts of late stage were higher than early stage. Isozyme bands of crude extracts samplinged in all habitats and all growing stages showed two bands and the signal intensity showed strongly according to passage of growing stage. The purified extracts of LH-4 fraction showed excellent inhibition effect in proliferation of HL-60 cells and markedly suppressed colony formation in mouse fibroblast cells. Dose response between partially purified extracts(400ppm) and negative control did not produced statistically significant reduction in colony formation.

• The Journal of Korean Academy of Prosthodontics
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• v.44 no.2
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• pp.250-263
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• 2006

Statement of problem: Ti-alloy has been used widely since it was produced in the United States in 1947 because it has high biocompatibility and anticorrosive characteristics. Purpose: The pure titanium, however, was used limitedly due to insufficient mechanical charateristics and difficult manufacturing process. Our previous study was focused on the development of a new titanium alloy. In the previous study we found that the Ti-Ta-Nb alloy had better mechanical characteristics and similar anticorrosive characteristics to Ti-6Al-4V Material and methods: In this study, the cytotoxicity of the Ti-Ta-Nb alloy was evaluated by MTT assay using MSCs(Mesenchaimal stem cells) and L929 cells(fibroblast cell line). The biocompatibility of the Ti-Ta-Nb alloy was performed by inserting the alloy into the femur of the rabbits and observing the radiological and histological changes surrounding the alloy implant. Results: 1. In the cytotoxicity test using MSCs, the 60% survival rate was observed in pure titanium, 84% in Ti-6Al-4V alloy and 95% in Ti-10Ta-10Nb alloy. 2. In the animal study, the serial follow-up of the radiographs showed no separation or migration revealing gradual bone ingrowth surrounding the implants. Similar radiographic results were obtained among three implant groups pure titanium, Ti-6Al-4V alloy and Ti-10Ta-10Nb alloy. 3. In the histologic examination of the bone block containing the implants. the bone ingrowth was prominent around the implants with the lapse of time. There was no signs of any tissue rejection, degeneration, or inflammation. Active bone ingrowth was observed around the implants. In the comparison of the three groups, the rate of bone ingrowth was better in the Ti-10Ta-10Nb alloy group than those in pure titanium group or Ti-6Al-4V alloy group. In conclusion, Ti-10Ta-10Nb alloy revealed better biocompatibility in survival rate of the cells and bone ingrowth around the implants. Therefore we believe a newly developed Ti-10Ta-10Nb alloy can replace currently used Ti-6Al-4V alloy to increase biocompatibility and to decrease side effects. Conclusion: In conclusion, Ti-10Ta-10Nb alloy revealed better biocompatibility in survival rate of the cells and bone ingrowth around the implants. Therefore we believe a newly developed Ti-10Ta-10Nb alloy can replace currently used Ti-6Al-4V alloy to increase biocompatibility and to decrease side effects.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.45 no.1
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• pp.49-56
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• 2019

Pueraria thomsonii Benth. as a medicinal ingredient, has been traditionally used in Chinese medicine to treat fever, acute dysentery, diarrhea, diabetes, and cardiovascular disease. The effects of P. thomsonii flower on skin have not been reported yet. In this study, the whitening effect of P. thomsonii flower was verified using B16F1 melanoma cells and HS68 fibroblasts. P. thomsonii flower extract reduced melanin contents of B16F1 cells in a dose-dependent manner. To identify its active components, we analyzed P. thomsonii flower extract using high performance liquid chromatography (HPLC). As a result, we identified three major isoflavones of tectorigenin, tectoridin, and tectorigenin 7-O-xylosylglucoside. At a non-cytotoxic concentration, the three components also reduced melanin contents of B16F1 cells in a dose-dependent manner. The depigmentation effects were attributed to the reduced gene expression of tyrosinase and microphthalmia-associated transcription factor (MITF). In order to elucidate another depigmentation mechanism, their effects on DKK-1, a fibroblast-derived depigmentation factor, was determined in HS68 cells. As a result, P. thomsonii flower extracts, tectoridin and tectorigenin 7-O-xylosylglucoside, reduced DKK-1 gene expression, while tectorigenin increased DKK-1 gene expression in a dose-dependent manner. These results suggest that tectorigenin can be used as an effective whitening agent that inhibit melanin synthesis in melanocytes and promote the secretion of depigmentation factor from fibroblasts.