• Journal of Embryo Transfer
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• v.16 no.3
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• pp.213-221
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• 2001

This study was conducted to examine in vitro developmental ability of porcine embryos after somatic cell nuclear transfer. The porcine ear fell was cultured in vitro for confluency in serum-starvation condition(TCM-199 + 0.5% FBS) far 3~6 days of cell confluency. The zona pellucida of IVM oocytes were partially drilled using laser system. Single somatic cell was individually transferred into enucleated oocytes. And the reconstructed embryos were electrically fused(single DC 1.9kv/cm, 30$\mu$ sec) with 0.3M mannitol. After electrofusion, embryos were activated(single AC 5v/mm, 5sec) and cultured in HCSU-23 medium containing 10% FBS at 39$^{\circ}C$, 5% $CO_2$ in air for 6 to 8 days. The fusion rate of donor cells was 45.6, 36.8 and 46.1% in 3~4, 5~6 days of serum starvation and non serum starvation(N-S), and were 52.7. 53.0 and 51.7% in 1~2. 5~6 and 13~14 passages of donor cell culture, respectively. No significant difference was found in the fusion rate of donor cells by the duration of serum starvation treatment or the number of donor cell passages. By the size of donor cells, however, the fusion rate was significantly higher(P<0.05) for reconstructed embryos derived from 25r $\mu$m $\geq$ site of donor cells (65.3%) than that of 25~30$\mu$ m(42.5%) or 30$\mu$ m(45.5%)$\leq$ cells. The cleavage rate was significantly (P<0.05) higher in 3~4 darts of serum starvation treatment(67.1%) than that in N-S (50.7%) or 5~6 days of starvation(57.1%). The activation rate by the size of donor cells in fused oocytes was 56.5, 68.8 and 58.5%, respectively, and was not significant.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.1-12
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• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• Korean Journal of Environmental Agriculture
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• v.24 no.2
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• pp.106-116
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• 2005

This experiment was conducted to investigate available extraction capacity and potential mobility of heavy metal according to the distribution property and contamination level of heavy metals in soils and to suggest a reform measure of soil environment assessment methodology applied with soil quality and the official soil heavy metal test methods in domestic and foreign countries. The soils were collected from the natural forest paddy with long-term application of same type fertilizer, and paddies near metal mine and industrial complex. The post-treatment methods of soil were partial extraction, acid digestion and sequential extraction methods. For the heavy metal contents with different soil properties, it was shown that their natural forest and paddy soil were slightly low and similar to the general paddy soil, while their paddies near metal mine and industrial complex were higher than the standard level of Soil Environment Protection Act. Heavy metal concentrations in the soils with different soil properties had difference between $HNO_3\;and\;HNO_3+HCl$ extractant by US-EPA 3051a method. There were highly significant positive relationships in both two methods. It was appeared that the higher extractable concentration ratio with 0.1N-HCl to total heavy metal content with $HNO_3+HCl$ extractant the greater total heavy metal content. There were highly significant positive correlationship between total heavy metal content and extractable content with 0.1N-HCl. For extractable capacity of soil extractable solution compared to the total heavy metal content it was appeared that it extractable method with 0.1N-HCl was higher than those with EDTA and DTPA. In extractable ratio with 0.1N-HCl in the contaminated paddy soils near mine and industrial complex, it was shown that the lower soil pH, the higher total heavy metal content. The order of a potential mobility coefficient by distribution of heavy metal content with ie different typies in the soil was Cd>Ni>Zn>Cu>Pb. It could be known that contamination characteristics of heavy metals with different types of soils were affected by different heavy metal components, contamination degree and soil chemical properties, and heavy metal concentration with different extractable methods had great variations with adjacent environment. To be compared with assessment methodology of soil environment impact at domestic and foreign countries with our results, it might be considered that there was necessary to make a single analysis method based on total heavy metal content with environmental overloading concept because of various analysis methods for total heavy metal content and present analysis method with great variation according to soil environment. In spite of showing higher concentration of heavy metal with acidic digestion than the extractable method, it might be considered that there is need to be adjusted the national standard of soil heavy metal contamination.

• Tuberculosis and Respiratory Diseases
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• v.43 no.5
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• pp.728-735
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• 1996

Background : Recognition and ingestion of opsonized microorganisms by neutrophils induces the burst of oxidative metabolic activity. Products of the respiratory burst activity provide powerful oxygen dependent killing mechanism. Measurement of respiratory burst activity has been a major indicator of the functional capacity of neutrophils. We determined the respiratory burst activity of neutrophils in patients with pneumonia and observed the changes during the clinical course of pneumonia. Methods: The EDTA blood was drawn from 24 normal controls and same numbers of pneumonia patients. The respiratory burst activity(with the production of $H_2O_2$ which changes nonfluorescent DCF-DA to green fluorescent DCF) in the non-stimulated state and the stimulated state with fMLP and PMA of neutrophils was measured by flowcytometry at day 1, 3, 5, 7 and 9 of admission. Results: The respiratory burst activity of neutrophils was mildly increased by stimulation with fMLP. But there was no statistical significance between normal control and patients with pneumonia. The respiratory burst activity of neutrophils was markedly increased by stimulation with PMA in both groups. There was a significant difference in response to PMA between normal control and patients with pneumonia. The production of hydrogen peroxide from neutrophils was decreased during early course of pneumonia and it was recuperated gradually to normal level in 9 days. Conclusion : Hydrogen peroxide production from neutrophils was suppressed during early course of pneumonia and restored after treatment. It is suggested that the production of oxygen radical in response to PMA stimulation from each neutrophils is decreased rather than increased during the early course of pneumonia.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.49 no.4
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• pp.313-321
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• 2023

Platycodon grandiflorus (P. grandiflorus) flower is a perennial plant belonging to the family Campanulaceae and has many excellent pharmacological effects, so it has been used as a medicinal ingredient since ancient times. In addition, anthocyanin is a purple or blue natural pigment contained in plant flowers and fruits, and is known as a powerful antioxidant. The purpose of this study was to confirm the dermatological functionality of P. grandiflorus flower extract and the value of the bluish anthocyanin contained in flowers as a cosmetic material as a natural pigment. Firstly, 50% ethanol and 80% ethanol were added to the P. grandiflorus flower and extracted under reflux for 4 h at 25, 60, and 80 ℃, and the pH of each treatment group was similar. Based on the anthocyanin content and chromaticity (E^*ab), 50% ethanol 60 ℃ extraction conditions showing the color development most similar to the natural color of the P. grandifloras flower were selected, and a sample was prepared by concentrating and lyophilizing. The analysis results showed that the total phenol, total flavonoid, and total anthocyanin contents were in the ranges of 23 ㎍/mL, 16 ㎍/mL, and 0.17 ㎍/mL, respectively. The P. grandiflorus flower extract suppressed the production of nitric oxide (NO) and interleukin-6 (IL-6) in lipopolysaccharide (LPS) induced RAW264.7 cells. Furthermore, the P. grandiflorus flower extract showed wound healing effects through the promotion of skin cell migration in TNF-α stimulated human keratinocytes. The stability of anthocyanin and extract color was studied during a storage period of 50 days at various temperatures (4 ℃, 25 ℃, and 45 ℃). Color values (L, a, and b) of the P. grandiflorus flower extract changed over 50 days, whereas the bluish-purple color of the extract was stabilized using 5% maltodextrin. These results suggest that P. grandiflorus flower extract may be useful as a natural cosmetic pigment.

• The Korean Journal of Nuclear Medicine Technology
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• v.25 no.2
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• pp.35-40
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• 2021

Purpose Glomerular filtration rate(GFR) is an important indicator for the diagnosis, treatment, and follow-up of kidney disease and is also used by healthy individuals for drug use and evaluating kidney function in donors. The gold standard method of the GFR test is to measure by continuously injecting the inulin which is extrinsic marker, but it takes a long time and the test method is complicated. so, the method of measuring the serum concentration of creatinine is used. Estimated glomerular filtration rate (eGFR) is used instead. However, creatinine is known to be affected by age, gender, muscle mass, etc. eGFR formulas that are currently used include the Cockroft-Gault formula, the modification of diet in renal disease (MDRD) formula, and the chronic kidney disease epidemilogy collaboration (CKD-EPI) formula for adults. For children, the Schwartz formula is used. Measurement of GFR using ⁵¹Cr-EDTA (diethylenetriamine tetraacetic acid), ^99mTc-DTPA (diethylenetriamine pentaacetic acid) can replace inulin and is currently in use. Therefore, We compared the GFR measured using ^99mTc-DTPA with the eGFR using CKD-EPI formula. Materials and Methods For 200 kidney transplant donors who visited Asan medical center.(96 males, 104 females, 47.3 years ± 12.7 years old) GFR was measured using plasma(Two-plasma-sample-method, TPSM) obtained by intravenous administration of ^99mTc-DTPA(0.5mCi, 18.5 MBq). eGFR was derived using CKD-EPI formula based on serum creatinine concentration. Results GFR average measured using ^99mTc-DTPA for 200 kidney transplant donors is 97.27±19.46(ml/min/1.73m²), and the eGFR average value using the CKD-EPI formula is 96.84±17.74(ml/min/1.73m²), The concentration of serum creatinine is 0.84±0.39(mg/dL). Regression formula of ^99mTc-DTPA GFR for serum creatinine-based eGFR was Y = 0.5073X + 48.186, and the correlation coefficient was 0.698 (P<0.01). Difference (%) was 1.52±18.28. Conclusion The correlation coefficient between the ^99mTc-DTPA and the eGFR derived on serum creatinine concentration was confirmed to be moderate. This is estimated that eGFR is affected by external factors such as age, gender, and muscle mass and use of formulas made for kidney disease patients. By using ^99mTc-DTPA, we can provide reliable GFR results, which is used for diagnosis, treatment and observation of kidney disease, and kidney evaluation of kidney transplant patients.

• Journal of Periodontal and Implant Science
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• v.24 no.3
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• pp.581-596
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• 1994

This in vitro study was undertaken to obtain optimal tetracycline concentration that aids proliferation and spreading of human periodontal ligament cells, for clinical application in root surfaces of periodontally diseased teeth. Periodontal ligament cells used in this study were obtained from explants of periodontal ligament of 1st premolar teeth which were extracted for the purpose of orthodontic treatment. The cells were cultured in Dulbecco's Modified Eagle Medium(DMEM) supplemented with 100 U/ml penicillin, $100\;{\mu}g/ml$ streptomycin and 10% FBS at $37^{\circ}C$, 100% humidity, 5% $CO_2-95%$ air. Cells were used between the third to 4th passage. After root planing of periodontally extracted teeth, the root slabs were cut with carborundum disk. In the cell proliferation experiment, experimental groups were root planing only group, immersed groups in 25, 50, 75, 100, 150mg/ml aqueous solution of Tetracycline HCl followed by a vigorous rinse in PBS. Human PDL cells at concentration of $1{\times}10^5\;cells/ml$ were seeded in each culture well which contained root slabs and incubated for 6 hours. Then, all of the root slabs were moved into new 24 culture well and incubated 24, 48 and 72 hours. The cell counting was done by inverted phase contrast microscope after trypsinization. The following results were obtained. The cell number was increased in order root planing only group, 25, 150, 50, 75, 100mg/ml of Tetracycline HCl treated group in 24, 48 and 72 hours. The maximal cell number was obtained when the root slabs were immersed in solution with 100mg/ml of Tetracycline HCl. There were statistically significant between the root planing only group and 75, 100 mg/ml of Tetracycline HCl treated group in 24 hours, between the root planing only group and 100mg/ml of Tetracycline HCl treated group in 48 hours, between the root planing only group and 50, 75, 100mg/ml of Tetracycline HCl treated group, between 25 and 100mg/ml of Tetracycline HCl treated group in 72 hours(p<0.05). In the cell spreading experiment, after 30 minutes of incubated, in the root planing only group, the cells were generally round in shape. The cell surface was mostly covered with blebs. The cells started to attach to root surface by cytoplasmic extension in 50, 100mg/ml of Tetracycline HCl treated groups, more numerous cells attached to root surface than root planing only group. Many orifices of dentinal tubule were exposed, cells showed radially spreaded cytoplasm and unspreaded central region of the cell was covered with blebs. After 6 hours of incubation, in the root planing only group, cells showed radially spreaded cytoplasm and were attached flat appearance. In 50, 100mg/ml of Tetracycline HCl treated groups, cellular margin was concaved and cytoplasm showed elongated appearance with polarity. After 24 hours of incubation, in the root planing group, cells showed characteristic polarity. In 50, 100mg/ml of Tetracycline HCl treated groups, cells showed more elongated and spindle - like appearance.