• Title/Summary/Keyword: In-vitro study

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Antigen analysis of Toxoplasma gondii Iysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot (EITB) (효소면역 전기영동이적법에 의한 톡소포자충 용해물 및 분비 항원의 분석)

  • 안명희;손혁진
    • Parasites, Hosts and Diseases
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    • v.32 no.4
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    • pp.249-258
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    • 1994
  • Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplosmo Iysate (RH strain) , frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain) . Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1 :512 of 10 days after primary immunization, 1 : 2,048 of 10 days after secondary immunization, 1: 1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplcsma major antigens corresponding to n of 24 kDa, 27 kDa,30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T gondii, the IgG antibody bud of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory- secretory materials and Iysate was important major antigen of T gondii (RH).

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Anti-inflammatory Effect of Lactuca sativa L. Extract in Human Umbilical Vein Endothelial Cells and Improvement of Lipid Levels in Mice Fed a High-fat Diet (상추 추출물(Lactuca sativa L.)의 혈관내피세포에서 항염증 작용과 고지방 식이 생쥐에서 혈중 지질농도 개선에 미치는 영향)

  • Hwang-Bo, Jeon;Jang, Kyung Ok;Chung, Hayoung;Park, Jong-Hwa;Lee, Tae Hoon;Kim, Jiyoung;Chung, In Sik
    • The Korean Journal of Food And Nutrition
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    • v.29 no.6
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    • pp.998-1007
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    • 2016
  • The objective of this study was to investigate the effects of a lettuce (Lactuca sativa L.) extract on the inflammation of human umbilical vein endothelial cell (HUVEC) and blood lipid improvement in hypercholesterolemic mice fed a high cholesterol diet. The lettuce extract (100% ethanol extract) inhibited the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in HUVEC treated with tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$). The lettuce extract suppressed the adhesion of THP-1 to TNF-${\alpha}$-treated HUVEC. The lettuce extract decreased the TNF-${\alpha}$-stimulated production of proinflammatory cytokine interleukin-6, interleukin-8 and chemokine monocyte chemotactic protein 1. In hypercholesterolemic mice, the lettuce extract reduced serum total cholesterol, triglyceride, and low-density lipoprotein-cholesterol level, while the lettuce extract elevated high-density lipoprotein-cholesterol level, resulting in the decrease of atherogenic index and cardiac risk factor level. These results suggested that lettuce extract can be an useful resource to show an anti-inflammatory effect and improve lipid metabolism.

Effect of Early Harvest on the Forage Yield, Quality and Regrowth of Italian Ryegrass and Barley Sown in Early Spring (이탈리안 라이그라스와 청보리 춘파 재배에서 조기 수확이 수량과 사료가치 및 재생에 미치는 영향)

  • Seo, Sung;Kim, Won-Ho;Kim, Ki-Yong;Jung, Min-Woong;Choi, Gi-Jun;Park, Hyung-Soo;Lee, Joung-Kyong
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.31 no.1
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    • pp.39-46
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    • 2011
  • This study was carried out to determine the forage yield, quality, and regrowth of Italian ryegrass (IRG) and barley sown on 2 March 2009 in Suwon. The five treatments were two IRG cultivars (Kowinearly with early maturity and Kowinmaster with medium maturity), one barley cultivar (Yuyeon), and two mixtures (Kowinearly + Yuyeon and Kowinmaster + Yuyeon). The first harvest date was 26 May, which was at late heading, heading and early dough stage of Kowinearly, Kowinmaster and barley, respectively. Regrowth yield was investigated on 29 June. The heading dates of Kowinearly and Kowinmaster were 16 May and 22 May, respectively, and that of barley was 13 May. The dry matter (DM) percentage were 13.0~18.4% at first harvest, and 22.5~24.8% at regrowth in all treatments. The forage yield of barley and Kowinmaster + Yuyeon mixture at first harvest was higher than that of IRG (p<0.05), but higher regrowth yield was observed in IRG, and then IRG + barley mixtures (p<0.05). The crude protein (CP) content and in vitro DM digestibility (IVDMD) of IRG at first harvest were 16.7~17.1% and 78.3~80.4%, respectively, which were higher than those of barley (CP 12.2% and IVDMD 72.6%) and IRG + barley mixtures. The total yields of DM, CP and digestible DM were high in Kowinmaster + Yuyeon mixture as 11,628 kg, 1,669 kg and 8,457 kg per ha, respectively. In conclusion, spring seeding of IRG + barley mixtures and/or barley were recommended when early harvest. Regrowth of IRG sown in early spring was vigorous. Mixture cultivation of IRG and barley was effective, because of forage yield and stable production, and harvest at June instead of May was desirable for forage productivity of spring sown IRG and barley.

Estrogeicity of Genistein and Bisphenol A (콩류식품의 주성분인 Genistein과 식품포장재 및 용기에 사용되는 Bisphenol A의 에스트로젠 효과에 관한 연구)

  • 강경선;이영순;신광순
    • Journal of Food Hygiene and Safety
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    • v.13 no.2
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    • pp.106-111
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    • 1998
  • This study has been focused on both estrogenic and proliferating activity of genistein (GEN) and bisphenol A (BPA). GEN and BPA enhance the proliferation of estrogen-dependent MCF-7 human breast cancer cells at concentrations as low as 100 nM of GEN and 8 ng/ml of BP A achieving similar effect to that of estradiol at 1 nM. Expression of the estrogen responsive gene, pS2 was also induced in MCF-7 cells by treatment with genistein at dose as low as 1 nM and BPA at dose as low as 4 ng/ml. Using 21 day-old ovariectomized nude mice, we examined end-bud formation and mammary gland development after treatment with bisphenol A or genistein. Compared with untreated control, mammary gland development and end-bud formation were significantly increased in mice fed genistein or bisphenol A (p<0.05). Taken together, it is concluded that GEN and BP A can act as an estrogen agonist resulting in cell proliferation and induction of the estrogen responsive pS2 gene in MCF-7 cells in vitro and in athymic mice in vivo, respectively. Therefore, it is suggested that GEN and BP A might modulate human endocrine system and these compounds might be considered as a endocrine modulator at the low levels of doses.

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EFFECT OF LIGHT IRRADIATION MODES ON THE MARGINAL LEAKAGE OF COMPOSITE RESIN RESTORATION (광조사 방식이 복합레진 수복물의 변연누출에 미치는 영향)

  • 박은숙;김기옥;김성교
    • Restorative Dentistry and Endodontics
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    • v.26 no.4
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    • pp.263-272
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    • 2001
  • The aim of this study was to investigate the influence of four different light curing modes on the marginal leakage of Class V composite resin restoration. Eighty extracted human premolars were used. Wedge-shaped class Y cavities were prepared on the buccal surface of the tooth with high-speed diamond bur without bevel. The cavities were positioned half of the cavity above and half beyond the cemento-enamel junction. The depth, height, and width of the cavity were 2 mm, 3 mm and 2 mm respectively. The specimens were divided into 4 groups of 20 teeth each. All the specimen cavities were treated with Prime & Bond$^{R}$ NT dental adhesive system (Dentsply DeTrey GmbH, Germany) according to the manufacturer's instructions and cured for 10 seconds except group VI which were cured for 3 seconds. All the cavities were restored with resin composite Spectrum$^{TM}$ TPH A2 (Dentsply DeTrey GmbH, Germany) in a bulk. Resin composites were light-cured under 4 different modes. A regular intensity group (600 mW/${cm}^2$, group I) was irradiated for 30 s, a low intensity group (300 mW/${cm}^2$, group II) for 60 s and a ultra-high intensity group (1930 mW/${cm}^2$, group IV) for 3 s. A pulse-delay group (group III) was irradiated with 400 mW/${cm}^2$ for 2 s followed by 800 mW/${cm}^2$ for 10 s after 5 minutes delay. The Spectrum$^{TM}$ 800 (Dentsply DeTrey GmbH, Germany) light-curing units were used for groups I, II and III and Apollo 95E (DMD, U.S.A.) was used for group IV. The composite resin specimens were finished and polished immediately after light curing except group III which were finished and polished during delaying time. Specimens were stored in a physiologic saline solution at 37$^{\circ}C$ for 24 hours. After thermocycling (500$\times$, 5-55$^{\circ}C$), all teeth were covered with nail varnish up to 0.5 mm from the margins of the restorations, immersed in 37$^{\circ}C$, 2% methylene blue solution for 24 hours, and rinsed with tap water for 24 hours. After embedding in clear resin, the specimens were sectioned with a water-cooled diamond saw (Isomet$^{TM}$, Buehler Co., Lake Bluff, IL, U.S.A.) along the longitudinal axis of the tooth so as to pass the center of the restorations. The cut surfaces were examined under a stereomicroscope (SZ-PT Olympus, Japan) at ${\times}$25 magnification, and the images were captured with a CCD camera (GP-KR222, Panasonic, Japan) and stored in a computer with Studio Grabber program. Dye penetration depth at the restoration/dentin and the restoration/enamel interfaces was measured as a rate of the entire depth of the restoration using a software (Scion image, Scion Corp., U.S.A.) The data were analysed statistically using One-way ANOVA and Tukey's method. The results were as follows : 1. Pulse-Delay group did not show any significant difference in dye penetration rate from other groups at enamel and dentin margins (p>0.05) 2. At dentin margin, ultra-high intensity group showed significantly higher dye penetration rate than both regular intensity group and low intensity group (p<0.05). 3. At enamel margin, there were no statistically significant difference among four groups (p>0.05). 4. Dentin margin showed significantly higher dye penetration rate than enamel margin in all groups (p<0.05).

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IN VITRO COMPARISON OF VARIOUS DIAGNOSTIC METHODS OF OCCLUSAL CAR10US LESIONS (교합면 우식병소의 다양한 진단법에 관한 비교연구)

  • Kim, Jae-Gon;Kim, Young-Jin;Kim, Young-Sin;Baik, Byeong-Ju
    • Journal of the korean academy of Pediatric Dentistry
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    • v.28 no.4
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    • pp.613-619
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    • 2001
  • The aims of this study were to compare the accuracy, sensitivity and specificity of cnventional visual examination, radiography and a new laser fluorescence method, KaVo Diagnodent, for the detection of occlusal caries lesions. One hundred sound human premolars and molars which had no restorations or interproximal cavities were tested by three methods. Tooth lesions depth was assessed at histologic examination using Caries detector dye The following results were obtained. 1. Diagnodent show 7.8 in sound tooth, 25.4 in initial caries, 30.5 in enamel caries, and 53.8 in dentin caries with average score 2. Spearman and Pearson relation coefficient was high between tooth-specimen test with dye and Diagnodent(0.736, 0.619), visual examination(0.664, 0.666), and was low between tooth-specimen test with dye and radiographic examination(P<0.01, total) 3. Accuracy of occlusal caries was highest on Diagnodent(65%) and lowest on radiographic examination(35%) 4. In initial caries, the sensitivity and specificity of Diagnodent method was the highest. In enamel caries, the sensitivity of visual examination was the highest and specificity of Diagnodent method was the highest. In dentinal caries, the sensitivity and specificity of Diagnodent method was the highest and sensitivity of visual examination was the lowest.

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Role of HOXA Gene in Human Endometrial Decidualization (인간 자궁내막의 탈락막화에서 HOXA10 유전자의 역할)

  • Lee, Chang-Se;Park, Dong-Wook;Park, Chan-Woo;Kim, Tae-Jin
    • Clinical and Experimental Reproductive Medicine
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    • v.37 no.3
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    • pp.207-216
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    • 2010
  • Objective: This study was performed to clarify the role of HomeoboxA (HOXA) and its related signaling molecules in the decidualization of primary cultured endometrial cells. Methods: Human endometrial tissues were obtained by curettage of hysterectomy specimens from patients with conditions other than endometrial diseases. Tissues were minced and digested with Trypsin-EDTA for 20 min, $37^{\circ}C$. Cells were cultured with DMEM/F12 medium in $37^{\circ}C$, 5% $CO_2$ incubator for 24 hrs. Cells were treated with HOXA10 siRNA and added transforming growth factor (TGF)-${\beta}1$ (10 ng/mL) for 48 hrs to induces decidualization in vitro. Reverse transcription polymerase chain reaction analysis was accomplished to observe the expression of HOXA10, prolactin, cyclooxygenase (COX)-2, peroxisome proliferatoractivated receptor (PPAR)-$\gamma$, and wingless-type MMTV integration site family (Wnt). Results: HOXA10 expression was increased (1.8 fold vs. non-treated control) in TGF-${\beta}1$ treated cells. Decidualization marker, prolactin, was significantly increased in TGF-${\beta}1$ treated cells compared with HOXA10 siRNA treated cells. Endometrial cell differentiation marker, COX-2 was down-regulated by HOXA10 siRNA even if cells were treated with TGF-${\beta}1$. Wnt4 was down-regulated by treated with HOXA10 siRNA, this expression patters was not changed by TGF-${\beta}1$. Expression of PPAR-$\gamma$ was down regulated by TGF-${\beta}1$ in regardless of HOXA10 siRNA treatment. Conclusion: TGF-${\beta}1$ which is induced by progesterone in endometrial epithelial cells may induces stromal cell decidualization via HOXA10 and Wnt signaling cascade.

Phospholipase C-γ Activation by Direct Interaction with β-Tubulin Isotypes (베타 튜불린에 의한 포스포리파제 C-감마1의 활성화)

  • Lee, In-Bum;Kim, Sung-Kuk;Choi, Jang-Hyun;Suh, Pann-Ghill;Chang, Jong-Soo
    • Journal of Life Science
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    • v.16 no.4
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    • pp.612-617
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    • 2006
  • Phosphoinositide-specific phospholipase $C-{\gamma}\;1\; (PLC-{\gamma}\;1)$ has pivotal roles in cellular signaling by producing second messengers, inositol 1,4,5-trisphosphate $(IP_3)$ and diacylglycerol (DG). Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of ${\alpha}-$ and ${\beta}-tubulin$ heterodimers in all eukaryotic cells. In humans, six ${\beta}-tubulin$ isotypes have been identified which display a distinct pattern of tissue expression. Previously we found that $PLC-{\gamma}\;1$ and one of four ${\beta}-tubulin$ isotypes including ${\beta}1$, ${\beta}2$, ${\beta}3$ and ${\beta}6$, colocalized in COS-7 cells and cotranslocated to the plasma membrane to activate $PLC-{\gamma}\;1$ upon agonist stimulation. In the present study, we demonstrate that the remaining two, tubulin ${\beta}4$ and ${\beta}5$, also showed a potential to activate $PLC-{\gamma}\;1$. The phosphatidylinositol 4,5-bisphosphate $(PIP_2)$ hydrolyzing activity of $PLC-{\gamma}\;1$ was substantially increased in the presence of purified ${\beta}4$ and ${\beta}5$ tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that tubulin ${\beta}4$ and ${\beta}5$ also activate $PLC-{\gamma}\;1$. Taken together, our results suggest that all the ${\beta}-tubulin$ isotype activates $PLC-{\gamma}\;1$ activity to regulate cellular signaling.

Antioxidant and Anti-Proliferative Activities of Rubus Fruits in Korea (국내산 나무딸기류 과일의 항산화 및 암세포 항증식 활성)

  • Jung, Hana;Lee, Hee Jae;Cho, Hyunnho;Hwang, Keum Taek
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.41 no.12
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    • pp.1649-1655
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    • 2012
  • This study was conducted to determine the polyphenols, flavonoids and antioxidant activity (FRAP) of the extracts (crushed by hand or a homogenizer) of Rubus fruits (blackberry, Korean raspberry, black raspberry, boysenberry and golden raspberry) produced in Korea. In addition, their nitric oxide (NO) scavenging activity in RAW 264.7 cells and anti-proliferative activity in HT-29 and KATO-3 cells were investigated. Polyphenol and flavonoid contents in the Rubus fruits ranged from 0.6 to 8.9 and from 0.1 to 7.9 mg/g fresh fruit, respectively. Black raspberry had the highest polyphenol and flavonoid contents among the Rubus fruits. The homogenized extracts of blackberry, Korean raspberry and golden raspberry fruits showed significantly higher polyphenol and FRAP values than the hand-crushed extracts. FRAP values of the Rubus fruit extracts were significantly correlated with their polyphenol (R=0.995) and flavonoid (R=0.967) contents. The Rubus fruit extracts suppressed the NO secretions in LPS-treated RAW264.7 cells. There were no significant differences between extracts obtained by crushing by hand and those obtained using a homogenizer. Proliferation rates of HT-29 and KATO-3 cancer cells treated with the Rubus fruit extracts at 0.1, 0.25 and 0.5 mg/mL were reduced by 3~32% and 0~57%, respectively. The homogenized extracts of blackberry and Korean raspberry fruits had significantly higher anti-proliferation activity against HT-29 cancer cells than the hand-crushed extracts. However, extraction method did not show any significant difference on proliferation of KATO-3 cancer cells. The NO scavenging activity of the Rubus fruit extracts were significantly correlated with the anti-proliferation activities of the HT-29 (R=0.602) and KATO-3 cells (R=0.498).

Free Radical Scavenging Effect and Oxidative Stress Protective Activity of Domestic Processed Polygoni Multiflori Radix (국내산 법제 하수오의 라디칼 소거능 및 산화적 스트레스 개선 효과)

  • Kim, Hyun Young;Kim, Jun Young;Cho, Eun Ju;Choi, Ji Myung;Hwang, Chung Eun;Lee, Hee Yul;Ahn, Min Ju;Lee, Jin Hwan;Kim, Yun-Geun;Ko, Keon Hee;Goo, Young-Min;Oh, Kyeong Yeol;Cho, Kye Man
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.44 no.6
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    • pp.809-815
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    • 2015
  • In this study, we confirmed biological compounds from methanol (MeOH) extract of processed Polygoni Multiflori Radix (PPMR), and the radical scavenging effect and oxidative stress protective activity of MeOH extract of PPMR were investigated under in vitro conditions using LLC-$PK_1$ renal epithelial cells. In HPLC analysis, MeOH extract of PPMR contained four species of biological compounds named 2,3,5,4'-tetrahydroxystilbene 2-O-${\beta}$-D-glucoside, emodin, chrysophanol, and rhein. 2,3,5,4'-Tetrahydroxystilbene 2-O-${\beta}$-D-glucoside was detected as the main compound in PPMR as 115.02 mg/kg. MeOH extract of PPMR showed 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS), and hydroxyl radical scavenging activities in a concentration- dependent manner. In particular, upon $50{\mu}g/mL$ of PPMR extract treatment, DPPH, ABTS, and hydroxyl radical scavenging activities were approximately 48.4%, 57.9%, and 81.2%, respectively. LLC-$PK_1$ cell viability declined in response to oxidative stress induced by pyrogallol, sodium nitroprusside (SNP), and morpholinosydnonimine (SIN-1) generators of NO, $O_2{^-}$, and $ONOO^-$, respectively. However, MeOH extract of PPMR significantly and dose-dependently inhibited oxidative-stressed LLC-$PK_1$ cell cytotoxicity. In fact, upon $50{\mu}g/mL$ of PPMR extract treatment, LLC-$PK_1$ cell viabilities were approximately 82.1%, 89.1%, and 77.6% compared to stress levels induced by pyrogallol, SNP, and SIN-1, respectively.