• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.6
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• pp.461-467
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• 2005

Background. 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3. Materials and methods. The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ using ${\alpha}$-minimal essential medium (${alpha}$-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 ${{\mu}M$. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability. Results. For concentration between 5 and 10 ${\mu}M$, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 ${\mu}M$. In the concentration of 15 ${\mu}M$, the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 ${\mu}M$ was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%. Conclusion. These results suggests an incubation period of 15 minutes at 15 ${\mu}M$ of CFSE provides best labelling of MC3T3 in vitro.

• Proceedings of the Korea Society for Energy Engineering kosee Conference
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• 1996.04a
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• pp.144-150
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• 1996

The Energy Efficiency Standards and Labelling Act, as amended by the Rational Energy Utilization Act, provides energy efficiency standards and labellings for six equipment categories(viz, refrigerators, air-conditioners, fluorescent lamps and ballasts, incandescent lamps, cars) authorizes the Ministry of Trade. Industry and Energy (MOTIE) to prescribe amended or new energy efficiency standards and labelling standards. This study was initiated by the KIER(Korea Institute of Energy Research) in 1992. KIER's assessment of the appliance energy efficiency standards is designed to evaluate their statistical and engineering analysis according to Korean Industrial Standards(KS). And to make didtinction between the poor efficiency and good efficiency models. 5 grades are classified depending on their tested energy efficiency. This year, based on our analysis, MOTIE mandated updated standards for refrigerators, air-conditioners, Incandescent lamps, and fluorescent lamps.

• Molecules and Cells
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• v.27 no.4
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• pp.391-396
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• 2009

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events - for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5$^{TM}$ and CyTRAK Orange$^{TM}$. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.

• Archives of Pharmacal Research
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• v.21 no.6
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• pp.651-656
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• 1998

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of aloesin in plasma was developed. After solid-phase extraction from plasma and derivatization of aloesin and compound AD-1, which was prepared from aloesin as a internal standard, with 9-anthroylnitrile in the presence of quinuclidine, the derivatives were separated on a Inertsil ODS-3 column using acetonitrile/methanol/water (3:1:6) as a mobile phase, and detected fluorimetrically at 460nm with excitation at 360nm. The detection limit of aloesin was 3.2ng/ml in plasma (S/N=3).

• The Journal of Korean Academy of Prosthodontics
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• v.33 no.2
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• pp.317-334
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• 1995

The purpose of this study was to compare the bone formation, maturation around HA-and titaniumcoated dental implants in dog. 5 hydroxyapatite coated IMZ implants and 5 titanium coated IMZ implants were placed into the previously extracted site in the mandible of 5 adults dogs. All dogs were injected intravenously Tetracycline, Alizalin red S, and Calcein for bone fluorescent labelling, After the experimental period of 16weeks, the dogs were sacrificed and tissue samples around the implants were obtained. Microscopic observations(ligth, polariged and fluorescence microscope), morphometric analysis, line profile with EPMA, and quantitative analysis for Ca,P, and Ti were performed. The results were as follows ; 1. Bone maturations around the implants were relatively lower than those of natural teeth. No significant differences in bone maturation and remodeling patterns were observed between the two implants groups. 2. Calcification of bone surrounding the implants was initiated in 8-11 weeks for HA-coated implants, while it took 11 weeks or more for Ti-coated implants. 3. Bone-to-implants contact ratio of 82.63% was recorded for HA-coated group and 72.25% for titanium coated group, with no significant difference between the two groups. 4. Bone around the implants exhibited reduced quantity of Ca and P in the $100{\mu}m$ region relative to natural teeth, while the rest of the regions showed no statistical differences. No significant differences were found between the two implant groups. 5. There was a separation of HA layer from the implant core and subsequent infiltration of inflammatory cells into the resulting space in the HA-coated implants, and evidences of phagocytosis of HA particles by macrophages. Bone calcification was more rapid around HA-coated implants compared to titanium-coated implants, but HA coated implants did not show any significant differences either in the degree of calcification or the bone-to-implant contact ratio over Ti coated implants. HA coated implants may have complications associated with HA absorption and separation of HA layer from the implant core.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.29 no.1
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• pp.119-147
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• 1999

The purpose of this study was to investigate the effects of the Co-60 γ irradiation on the osseointegration. 2.0 mm titanium alloy screw implants(Sankin Industry Co. Ltd., Japan) were placed in the tibial metaphysis of the rabbits, bilaterally. The mean length of the implants was 6.0 mm. The right tibia was irradiated with a single dose of 15Gy from 60Co teletherapic machine at 5th postoperative day. The experimental group was irradiated tibia. The control group was non­irradiated tibia. To observe the phase of bone formation, the bone labeling by intramuscular injection of 20mg/Kg of Tetracycline, Calcein, Alizarin red S, was performed. The rabbits were sacrificed on the 1st, 2nd, 4th, 6th, 8th week and the tibia including implants were taken, and then the specimens were examined by the microradiography, light microscopy, and fluorescent microscopy. The obtained results were as follows: 1. There were connective tissue between bone and titanium at 1st week, in both group. Especially, the many empty lacunae without nucleus and obscure cytoplasm in experimental group, were observed. 2. The osteons were observed at 4th week in control group, and at 6th week in experimental group. The bone formation in experimental group was retarded as compared to the control group. 3. In fluorescent microscopy, bone labelling band was observed as linear, arc or concentric shape. Occasionary interrupted labelling band was observed, which is demonstrated bone remodeling. 4. In microradiographic examination, the radiolucent image was found between bone and implant with widening of bone marrow spaces as compared to the control group.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.26 no.1
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• pp.59-72
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• 2000

In this study, the rate of bone formation and the pattern of bone to implant contact surface around HA coated implant and pure Ti implant inserted into the irradiated tibia of rabbit were compared. Sixteen mongrel mature male rabbits were used as experimental animal. Each rabbit received 15 Gy of irradiation. Four weeks after irradiation, two holes were prepared on the tibia of each rabbit for placement of HA coated type and pure Ti type implants. Prior to implant placement, one group received steroid irrigation and the control group was similarly irrigated with normal saline. This was immediately followed by placement of the two different types of implants. Postoperatively, tetracycline was injected intramuscularly for 3 days. For fluorescent labelling, 3 days of intramuscular alizarine red injection was given. 2 weeks before sacrifice, followed by intramuscular calcein green on the last 3 days before specimen collection. Each rabbit was sacrificed on the second, fourth, sixth and eighth week after the implantation. The specimens were observed by the light microscope and the fluorescent microscope. The results were as follows; 1. All implants inserted into the irradiated tibia of rabbit were free from clinical mobility and no signs of bony resorption were noted around the site of implant placement. 2. Under the light microscope, new bone formation proceeded faster around implants that received steroid irrigation compared to the control group irrigated with saline. Bone to implant contact surface was greater in the steroid irrigated group than the saline irrigated group. Therefore, better initial stabilization was observed in the group pretreated with steroid irrigation. 3. Under the light microscope. HA coated implants showed broader bone to implant contact surface than pure Ti implants, and HA coated implants had better bone healing pattern than pure Ti implants. 4. In the steroid pretreated group, acceleration of bone formation was demonstrated by fluorescent microscopy around the 2, 4 weeks group and the 6 weeks HA coated implant group. The difference in the rate of bone formation proved to be statistically significant(P<0.05). Faster bone formation was noted in the saline irrigated group in the 6 weeks pure Ti implants and 8 weeks group. The difference was not statistically significant(P<0.05). 5. For the rabbits that were sacrificed on the second and fourth week after the implant placements, the rates of bone formation around HA coated implants proceeded faster than those around pure Ti implants under the fluorescent microscopy. For the rabbits that were sacrificed on the sixth week after the implant placements, the rates of bone formation around pure Ti implants proceeded faster than those around HA coated implants under the fluorescent microscopy. But this result did not show statistical significance (P<0.05) For the rabbits that were sacrificed on the eighth week after the implant placements, the rates of bone formation around HA coated implants proceeded faster than those around pure Ti implants under the fluorescent microscopy. This result was statistically significant (P<0.05).

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.218-223
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• 1993

Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.3
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• pp.193-202
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• 1991

Antimicrobial substances were screened by paper disk plate method in marine sponges, Halichondria okadai, Halichendria sp., H iaponica and Haliclona Pemollis, collected from the south coast of Korea. Antibacterial components were detected in two species, H okadai and Halichondria sp.. Three components such as benzoic acid, okadaic acid(OA) and dinophysistoxin-1(DTX1) were identified from these sponges as the antimicrobial compounds by MS and NMR spectral data. OA$(550{\~}600{\mu}g/kg)$ and $(400{\~}490{\mu}g/kg)$ were determined from the wet H okadai and Halichondria sp., respectively, by using fluorometric HPLC analysis with 9-anthryldiazomethane(ADAM) as fluorescent labelling reagent.

• Restorative Dentistry and Endodontics
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• v.24 no.2
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• pp.286-298
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• 1999

The endodontic-periodontic combined lesions have been difficult to get correct diagnosis and predictable treatment. This study was to make the experimental endodontic-periodontic combined defects in dogs for the study of the periodontal regeneration and to evaluate the efficacy of the enamel matrix protein and e-PTFE membrane in the experimental endodontic-periodontic combined defects. 5 mongrel dogs were used. The pulp chambers were opened and the plaque was inserted into the chambers to induce the periapical lesions on the mandibular second, third and fourth premolars of the dogs. 1 month later, the root canal treatments were done with gutta perch a and ZOE sealer. On the day of surgery, the periapical defects were standardized by trephine bur. The buccal dehiscence defects were made by the dental bur and bone chisels. The apicoectomy with retrofilling was done. The prepared roots were randomly selected for test and control groups. In the experimental groups, the enamel matrix derivative and e-PTFE membrane were used. Nothing was placed on the control group. Fluroscent labelling was used to evaluate the bone formation. After 4 and 12 weeks, the dogs were sacrificed and undecalcified sections were prepared and stained with toluidine blue. Those histologic sections were examined by fluorescent microscopy and light microscopy. The results were as follows. 1. In the control group, new bone was formed in the periapical defects and scarcely in the buccal dehiscence defects. New cementum was not detected at 4 and 12 weeks. 2. In the experimental groups, new bone, new cementum and periodontal ligament were found in the periapical and buccal dehiscence defects. The relative amount and the quality of the new bone, new cementum and periodontal ligament tissue that had formed on the experimental groups were superior to those of the control group. 3. The current observation implicated that e-PTFE membrane and enamel matrix protein could be the effective tools for the guided tissue regeneration of the endo-perio combined defects.