• Journal of Ginseng Research
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• 제43권1호
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• pp.86-94
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• 2019

Background: Ginseng is believed to have antitumor activity. Autophagy is largely a prosurvival cellular process that is activated in response to cellular stressors, including cytotoxic chemotherapy; therefore, agents that inhibit autophagy can be used as chemosensitizers in cancer treatment. We examined the ability of Korean Red Ginseng extract (RGE) to prevent autophagic flux and to make hepatocellular carcinoma (HCC) cells become more sensitive to doxorubicin. Methods: The cytotoxic effects of total RGE or its saponin fraction (RGS) on HCC cells were examined by the lactate dehydrogenase assay in a dose- or time-dependent manner. The effect of RGE or RGS on autophagy was measured by analyzing microtubule-associated protein 1A/1B-light chain (LC)3-II expression and LC3 puncta formation in HCC cells. Late-stage autophagy suppression was tested using tandem-labeled green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3. Results: RGE markedly increased the amount of LC3-II, but green and red puncta in tandem-labeled GFP-mRFP-LC3 remained colocalized over time, indicating that RGE inhibited autophagy at a late stage. Suppression of autophagy through knockdown of key ATG genes increased doxorubicin-induced cell death, suggesting that autophagy induced by doxorubicin has a protective function in HCC. Finally, RGE and RGS markedly sensitized HCC cells, (but not normal liver cells), to doxorubicin-induced cell death. Conclusion: Our data suggest that inhibition of late-stage autophagic flux by RGE is important for its potentiation of doxorubicin-induced cancer cell death. Therapy combining RGE with doxorubicin could serve as an effective strategy in the treatment of HCC.

• 치과방사선
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• 제29권1호
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• pp.105-117
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• 1999

Purpose: Ionizing radiations have been reported as an apoptosis initiating stimulus in various cells and it has established that sustained elevations in [Ca/sup 2+/] can lead to DNA fragmentation by Ca/sup 2+/-dependent endonucleases, ultimately resulting in apoptotic cell death. The previous experiments have been reported by using primarily thymocytes and lymphocytes and the change of [Ca/sup 2+/] was measured only by minutes or hours respectively. We need to evaluate [Ca/sup 2+/] in both several minutes and hours after irradiation of radiation of radiation therapy and verify the apoptotic cells. Materials and Methods: We have measured [Ca/sup 2+/] in human gingival epitheloid cancer cell with 10Gy irradiation, at minutely intervals and hourly intervals using digitized video-intensified fluorescence microscopy and the fluorescent Ca/sup 2+/ indicator dye, fura-2. In order to find out that the transient rise in [Ca/sup 2+/] could induced apoptosis, cells were incubated for 1 hour at 37℃ with TdT enzyme, rinsed and resuspended containing fluorescence and observed under a confocal fluorescence microscope. MTT assay was done to determine cell activity and LDH assay was done to determine the amount of necrotic cells. Results: After irradiation, the transient and temporal increasing of [Ca/sup 2+/] in the KB cells was founded. Though, there was no change in the intracellular [Ca/sup 2+/] at 30 minutes and 2 hours after irradiation. We could detect of DNA fragmented cells at 4 hours after 10Gy irradiated cells. There were no significant differences between 4 hour, 1 day, 3 day cells. There were no significant differences in MTT and LDH assay between the irradiated group and the control group after 4 hours and 1 day. Though after 3 days there were differences in MTT and LDH assay between the irradiated group was significantly decreased than the control group, in LDH assay the number of necrotic cell death of the irradiated was higher than the control group. Conclusion: In KB cells there were incipient and temporal increasing of the [Ca/sup 2+/] with 10Gy irradiation and the apoptosis was founded from 4 hours later which was earlier than seeing of the change of the amount of the cellular ability and necrosis.

• 생명과학회지
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• 제17권12호
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• pp.1723-1728
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• 2007

본 연구에서는 다양한 기능성을 가지는 복분자를 80% methanol로 추출한 후 분획물들을 제조하여 항산화 효과 및 인간 유래 위암세포인 AGS와 KatoIII을 이용하여 성장 저해 효과와 DNA손상을 측정하였다. 항산화 효과를 측정해 보기 위하여 총 폴리페놀 함량과 플라보노이드 함량, DPPH free radical의 소거활성을 측정하였는데, 먼저 폴리페놀의 경우 butanol fr., ethylacetate fr., water fr.순으로 나타났고 특히 butanol fr.의 경우 $546.25{\mu}g/mg$의 매우 높은 폴리페놀 함량을 확인 할 수 있었다. 플라보노이드의 함량 결과 ethylacetate fr., butanol fr., water fr.,순으로 나타났으며 ethylacetate fr.의 경우 $141.78{\mu}g/mg$의 높은 플라보노이드 함량을 확인할 수 있었다. DPPH free radical의 소거활성에서는 폴리페놀 함량의 순서와 같은 효과를 보였으며, 합성 항산화제인 BHA의 $RC_{50}$값 $3{\mu}g/ml$보다 water fr.을 제외하고 더 좋은 소거능을 확인 할 수 있었다. 인간 유래 위암세포주의 성장 저해 효과를 살펴본 결과 AGS와 KatoIII 세포주 모두 butanol fr.에서 가장 좋은 저해능을 나타내었고, butanol fr., ethylacetate fr., water fr. 순서로 저해효과를 나타내었다. DNA손상을 측정한 결과 역시 이와 같은 순으로 나타났으며, butanol fr.에서 1시간 이내에 약 50%정도의 손상유도 효과를 보였다. 이상의 결과에서 복분자 methanol추출물과 각각의 분획물은 항산화 효과 및 위암 세포주에 대한 성장 저해 활성 및 높은 DNA손상 유도 효과를 갖고 있음을 알 수 있었다.

• 한국물환경학회지
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• 제26권2호
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• pp.261-267
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• 2010

In order to evaluate the distribution level of Cryptosporidium and Giardia in drinking water resources, distribution of Cryptosporidium and Giardia was studied in intake water of main water treatment facility with treatment capacity over 50,000 ton/day in Korea. 10 L samples from each study sites were collected quarterly for 2 years between Oct. '04 and Dec. '07. Cryptosporidium and Giardia were filtered and concentrated by capsule filter and centrifugation, and analyzed by immunomagnetic separation process and fluorescent assay. Cryptosporidium oocysts and Giardia cysts were detected in 7.0% and 9.3%, respectively, of a total 776 samples from 97 study sites. And mean detection number of Cryptosporidium oocysts and Giardia cysts in total 776 samples was 0.11/10 L, 0.16/10 L, detection range of Cryptosporidium oocysts and Giardia cysts was 0~7/10 L, 0~4/10 L, respectively. In seasonal distribution, Cryptosporidium was more frequent in spring as 9.2% than other season, Giardia was more frequent in winter as 14.6% than other season, but there was not shown significant seasonal characteristics. In correlation analysis with total 776 data, Cryptosporidium had significant correlation to total coliforms at the 0.05 level, but correlation value was too low as 0.07 (r=0.07). In case of Giardia, what had significant correlation at the 0.05 level was total coliforms and fecal coliforms, but correlation value was too low as 0.26, 0.27 respectively.

• 대한의생명과학회지
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• 제6권2호
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• pp.141-149
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• 2000

우리 나라에서 발생하고 있는 급성 출혈성 질환인 신증후출혈열의 원인 바이러스는 Family Bunyaviridae의 Genus Hantavirus에 속하는 한탄과 서울바이러스에 의하여 발생되고 있다. 본 연구에서는 신증후출혈열로 의뢰된 환자에서 한탄바이러스에 대한 항체가를 간접면역형광항체법(indirect immunofluorescent antibody technique, IFAT), 면역효소측정법 (enzyme-linked immunosorbent assay, ELISA) (IgG, IgM), 고비중입자응집반응 (high density composite particle agglutination, HDPA) 및 플라크감소중화시험 (plaque reduction neutralization test, PRNT) 등으로 비교 측정하였고, 신증후출혈열 환자로 확진된 15명의 한탄바이러스 혈청형을 PRNT와 혈청형 특이 역전사 효소 중합효소연쇄반응(nested reverse transcriptase polymerase chain reaction, nested RT-PCR)으로 확인하였다. 신증후출혈열로 의뢰된 환자에서의 한탄바이러스에 대한 IFAT, ELISA (IgG, IgM), HDPA그리고 PRNT비교에서 형광항체, ELISA IgG,응집항체 및 중화항체는 8명 모두 높게 나타났으며, ELISA IgM은 5명에서는 현저히 높은 항체를 보유하고 있었다. 신증후출혈 열 환자 15명에서는 높은 형광항체와 중화항체 역가를 나타내었고, 15명 중 12명은 한탄바이러스, 2명은 서울바이러스에 대한 높은 중화항체를 갖고 있었으며, 1명은 두 바이러스에 대하여 동일한 항체 역가를 나타내었으며, 혈청형 특이 primer를 사용한 nested RT-PCR에서는 15명 중 3명과 1명만이 한탄바이러스와 서울바이러스 primer에 대해 RNA가 검출되었다.

• 대한수의학회지
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• 제37권3호
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• pp.583-593
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• 1997

Indirect fluorescent antibody test(IFAT) and enzyme-linked imuunosorbent assay (IgG-ELISA) as serological diagnostic tools were conducted to evaluate the usefulness for diagnosis of canine babesiosis infected with Babesia gibsoni in domestic various dog breeds, american pit bullterrier, military shepherd, and mongrel dogs. The results obtained from this study were abstracted as follows. The nonionic detergent Triton X-100 and absorbent bio-bead $SM_2$ were useful reagents for the preparation of pure merozoite antigen of B gibsoni to be used in ELISA. The optimum reaction in ELISA was shown when the protein concentration of ELISA antigen was measured as 625ng/ml and the conjugate concentration was diluted into 1/6000 fold. The average OD value of ELISA in sera determined with negative responses in IFAT was measured as $0.255{\pm}0.051$(490nm) and the cut - off value of OD was determined as 0.399(490nm). The serum antibodies in both of IFAT and ELISA were detected on one week after artificially infected with B gibsoni and these high antibody titers, 512X in IFAT and 1024X in ELISA, were long lasted until 15 weeks after infection. The reproducibility of reaction and stability of the antigen absorbed microtitration polystyrene plate preserved in $4^{\circ}C$ refrigerator and $-20^{\circ}C$ freezer, respectively could be lasted until 135 days after storage. The positive rates in IFAT by dog breeds were shown 8.1%(60/744 heads) in mongrel dogs, 81.3%(78/96 heads) in american pit bullterrier and 15.6%(15/96 heads) in military shepherd, while the positive rate in ELISA shown 17.6%(131/744 heads) in mongrel dogs, 83.3%(80/96 heads) in american pit bullterrier and 36.5%(35/96 heads) in military shepherd, respiectively. In the total of 936 heads surveyed with IFAT and ELISA the positive rates in IFAT and ELISA were 16.4%(153/936 heads) and 26.3%(246/936 heads), respectivily. Agreement of reactions between IFAT and ELISA was shown 82.4% in 936 dog sera. The specificity and sensitivity of ELISA reaction were 83.5% and 76.5%, respectively. From the conclusion obtained in this study it was evaluated that IFAT and ELISA were useful as highly specific, sensitive and stable serelogical tools for the diagnosis of canine babesiosis in Korea.

• 대한화학회지
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• 제54권4호
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• pp.451-459
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• 2010

세포내의 중금속이온의 형광검출은 유기분자화학과 세포생물학분야에서 높은 관심을 갖는다. 이 연구는 형광화학센서(FS)를 이용한 Hg$^{2+}$ 과 Zn$^{2+}$의 세포 내 검출을 목적으로 하였다. FS는 약한 형광을 나타내지만 Zn$^{2+}$와 결합 시에는 강한 방출 형광은 낸다. 2FS/Zn$^{2+}$의 형광증가는 FS-Hg$^{2+}$결합의 형성할 때 Hg$^{2+}$ 1당량만 추가에도 완전한 형광감소를 나타낼 수 있었다. 네가지의 세포주(LLC-MK2, Hela, HT29 and AMC-HN3)는 공촛점 현미경에 대한 형광이미지를 위하여 사용하였다. 세포생존능은 LLC-MK2 세포주에 FS, Zn$^{2+}$, FS-Zn$^{2+}$, Hg$^{2+}$의 처리 후에 평가하였다. FS의 세포독성능은 80%이상의 생존능을 보였다. 본 연구에서는 FS가 살아있는 세포내의 Hg$^{2+}$과 Zn$^{2+}$의 선택적 이미지를 검출할 수 있음을 보여주었다.

• Journal of Ginseng Research
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• 제41권2호
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• pp.169-179
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• 2017

Background: Ginsenosides are the main pharmacological components of Panax ginseng root, which are thought to be primarily responsible for the suppressing effect on oxidative stress. Methods: 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and oxygen radical absorption capacity were applied to evaluate the antioxidant activities of the ginsenosides. Human embryonic kidney 293 (HEK-293) cells were incubated with ginsenosides extracted by pulsed electric field (PEF) and solvent cold soak extraction (SCSE) for 24 h and then the injury was induced by $40{\mu}M$ $H_2O_2$. The cell viability and surface morphology of HEK-293 cells were studied using MTS assay and scanning electron microscopy, respectively. Dichloro-dihydro-fluorescein diacetate fluorescent probe assay was used to measure the level of intracellular reactive oxygen species. The intracellular antioxidant activities of ginsenosides were evaluated by cellular antioxidant activity assay in HepG2 cells. Results: The PEF extracts displayed the higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and stronger oxygen radical absorption capacity (with an oxygen radical absorption capacity value of $14.48{\pm}4.04{\mu}M\;TE\;per\;{\mu}g/mL$). The HEK-293 cell model also suggested that the protective effect of PEF extracts was dose-dependently greater than SCSE extracts. Dichloro-dihydro-fluorescein diacetate assay further proved that PEF extracts are more active (8% higher than SCSE extracts) in reducing intracellular reactive oxygen species accumulation. In addition, scanning electron microscopy images showed that the HEK-293 cells, which were treated with PEF extracts, maintained more intact surface morphology. Cellular antioxidant activity values indicated that ginsenosides extracted by PEF had stronger cellular antioxidant activity than SCSE ginsenosides extracts. Conclusion: The present study demonstrated the antioxidative effect of ginsenosides extracted by PEF in vitro. Furthermore, rather than SCSE, PEF may be more useful as an alternative extraction technique for the extraction of ginsenosides with enhanced antioxidant activity.

• 한국가축번식학회지
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• 제9권2호
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• pp.133-139
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• 1985

An immunoassay may be defined as an analytical procedure involving the competitive reaction between a limiting concentration of specific antibody and two populations of antigen, one of which is labelled or immobillized. The advent of immunoassay has revolutionised our knowledge of reproductive physiology and the practice of veterinary and clinical medicine. Radioimmunoassay (RIA) was the first of these methods to be developed, which meausred the analyte with good sensitivity, accuracy and precision (1,2). The essential components of RIA are:-(i) a limited concentration of antibodies, (ii) a reference preparation, and (iii) an antigen labelled with a radioisotope (usually tritium or iodine-125). Most procedures invelove isolating the antibody-bound fraction and measuring the amount of labelled antigen. Good facilities are available for scintilltion counting, data reduction nd statistical analysis. RIA is undergoing refinement through:-(i) the introduction of new techniques to separate the antibody-bound and free fractions which minimize the misclassification of labelled antigen into these compartments, and the amount of non-specfic binding. (3), (ii) the development of non-extration for the measurement of haptens (4), (iii) the determination of a, pp.rent free (i.e. non-protein bound) analytes (5), and (iv) the use of monoclonal antibodies(6). In 1968, Miles and Hales introduced in important new type of immunoassay which they termed immunora-diometric assay (IRMA) based on t도 use of isotopically labelled specific antibodies(7) in a move from limited to excess reagent systems. The concept of two-site IRMAs (with a capture antibody on a solid-phase, and a second labelled antibody to a different antigenic determinant of the analyte) has enabled the development of more sensitive and less-time consuming methods for the measurement of protein hormones ovar wide concentration of analyte (8). The increasing use of isotopic methos for diverse a, pp.ications has exposed several problems. For example, the radioactive half-life and radiolysis of the labelled reagent limits assay sensitivity and imposes a time limit on the usefulness of a kit. In addition, the potential health hazards associated with the use and disposal of radioactive cmpounds and the solvents and photofluors necessary for liquid scientillation counting are incompatable with the development of extra-laboratory tests. To date, the most practical alternative labels to radioisotopes, for the measurement of analytes in a concentration > 1 ng/ml, are erythrocytes, polystyrene particiles, gold sols, dyes and enzymes or cofactors with a visual or colorimetric end-point(9). Increased sensitivity to<1 pg/ml may be obtained with fluorescent and chemiluminescent labels, or enzymes with a fluorometric, chemiluminometric or bioluminometric end-point. The sensitivity of any immunoassay or immunometric assay depends on the affinity of the antibody-antigen reaction, the specific activity of the label, the precision with which the reagents are manipulated and the nonspecific background signal (10). The sensitivity of a limited reagent system for the measurement of haptens or proteins is mainly dependent upon the affinity of the antibodies and the smalleest amount of reagent that may be manipulated. Consequently, it is difficult in practice to improve on the sensitivity obtained with iodine-125 as the label. Conversely, with excess reagent systems for the measurement of proteins it is theoretically possible to increase assay sensitivity at least 1000 fold with alternative luminescent labels. To date, a 10-fold improvement has been achieved, and attempts are being made to reduce the influence of other variables on the specific signal from the immunoreaction.

• 생명과학회지
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• 제20권4호
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• pp.519-527
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• 2010

Paclitaxel은 미세소관의 탈중합을 억제하는 시약으로 알려져 있다. Paclitaxel은 다양한 세포에서 세포 내 방추체를 안정화시킴으로써 유사분열 억제 및 세포사멸을 유도한다. 본 실험에서는 토끼 관절 연골세포에서 paclitaxel이 연골세포의 증식과 사멸에 미치는 효과에 대한 연구를 수행하였다. MTT assay를 수행한 결과 paclitaxel은 연골세포에서 농도 의존적으로 세포 증식을 억제한다는 것을 확인 할 수 있었으며, FACS analysis와 Western blot analysis를 수행한 결과, paclitaxel이 G2/M 정지를 유도하는 것을 확인하였다. 또한, paclitaxel이 비정상적인 세포 분열유도와 핵 단편분절 유도없이 일어나는 mitotic catastrophe 즉, caspase-3 비의존적인 세포사멸을 유도하였다. Paclitaxel을 처리한 세포에서 일어나는 이러한 mitotic catastrophe에 의한 세포 죽음은 G1/S기의 진행을 억제하는 시약인 thymidine을 처리하는 것에 의해 억제되는 것을 확인할 수 있었다. 이러한 결과를 종합해 볼 때, paclitaxel에 의한 토끼 관절 연골 세포에서의 세포 죽음은 caspase-3 비의존적인 mitotic catastrophe에 의해 일어나는 것으로 사료되어진다.