• Journal of Society of Preventive Korean Medicine
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• v.12 no.2
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• pp.197-206
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• 2008

The inhibition of osteoblast by glucocorticoid is recognized as its action mechanism of decreased bone formation. In this study, the effect of JY, Jahyulyangkeuntang, on the differentiation and mineralization of osteoblastic cells was investigated in the presence of dexamethasone. The cell counting, enzyme activity assay, MTT assay, collagen content assay were done to determine the cell proliferation, alkaline phosphatase(ALP) activity, bone martrix production, and cell apoptosis. JY enhanced the cell proliferation after the culture for 10 days. ALP activity and total protein synthesis, and intracellular collagen synthesis were increased when the cells were treated with JY. And JY restored calvarial cell function decreased by dexamethasone.

• BMB Reports
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• v.35 no.3
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• pp.262-266
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• 2002

A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-asociated HIV-p24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.3
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• pp.874-879
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• 2004

Alakaline phosphatase (ALP)- or horseradish peroxidase (HRP)-antibody conjugate was used frequently on the immunological detection methods such as enzyme-linked immunosobent assay (ELISA), immunobolt, immunohistochemistry. The classical enzyme-antibody coupling method by one-step (direction) injection of glutaraldehyde bring into being disadvantage such as low sensitivity of antigen detection because of homopolymers. This study was modified with the dialysis glutaraldehyde method to provide simple coupling through E-amino residues present in most protein. The dialysis glutaraldehyde coupling effects were better than the classical one-step glutaraldehyde injection in antigen detection of ELISA and immunobolt. Optimal dose of the dialysis glutaraldehyde solution was 0.10-0.25 %. This results suggest that the dialysis glutaraldehyde coupling method can readily applied to antigen detection of in vitro and in vivo.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.163-180
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• 2001

The aim of this study was to evaluate the effects of chitosan coating on the attachment, proliferation, functional and morphological change of periodontal ligament cells. Primary human periodontal ligament cells were cultured in dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. In experimental group, cells of 4th to 7th passage were inoculated in the multiwell plates coated with chitosan in concentration of 0.22, 0.2, and $2mg/m{\ell}$. Cell counting and MTT assay were done after 0.5, 1.5, 3, 6 and 24 hours of incubation to evaluate the cell attachment, and then after 2 and 7 days of culture to evaluate the cell proliferation. The alkaline phosphatase activity was measured after 4 and 7 days of culture and the ability to produce mineralized modules was evaluated after 21 days of culture. The results were as follows : 1. The morphology of periodontal ligament cells on the chitosan coating was round or spheric. Round cells were aggregated after 6 hours of culture. Aggregated cells on the chitosan coated surface showed nodule-like appearance after 24 hours of culture and not achieved confluency at 7 days. 2. During early period of culture, the attachment of periodontal ligament cells were inhibited by chitosan coating. Inhibition of cell attachment tended to increase with the concentration of chitosan. 3. At the chitosan concentration of 0.02 and $0.2mg/m{\ell}$, periodontal ligament cells were more rapidly proliferated at 7 days, compared to the control group. At the concentration of $2mg/m{\ell}$, the proliferation of periodontal ligament cells was inhibitied(p<0.01). 4. Alkaline phosphatase activity of periodontal ligament cells was increased in chitosan coated group, especially at the concentration of $0.02mg/m{\ell}$after 4 days of culture.5. Periodontal ligament cells produced mineralized nodules on chitosan coated wells without the addition of mineralized nodule forming materials (ascorbic acid, ${\beta}-glycerophosphat$, dexamethasone). With the addition of mineralized nodule forming materials, periodontal ligament cells produced more mineralized nodules at the concentration of $0.02mg/m{\ell}$, compared to the control. In summary, the attachment, proliferation, cell activity, and alkaline phosphatase activity of periodontal ligament cells depended on the concentration of coated chitosan. Chitosan stimulated mineralized nodule formation by periodontal ligament cells. At the appropriate concentration($0.02mg/m{\ell}$), chitosan could increase alkaline phosphatase activity and stimulate the formation of mineralized nodule by periodontal ligament cells. These results suggest that chitosan can be used as an adjunct for bone graft material, and the matrix of tissue engineering for periodontal regeneration, especially bone regeneration.

• Nutrition Research and Practice
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• v.10 no.2
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• pp.148-153
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• 2016

BACKGROUND/OBJECTIVES: Bone formation and bone resorption continuously occur in bone tissue to prevent the accumulation of old bone, this being called bone remodeling. Osteoblasts especially play a crucial role in bone formation through the differentiation and proliferation. Therefore, in this study, we investigated the effects of Scytosiphon lomentaria extract (SLE) on osteoblastic proliferation and differentiation in MC3T3-E1 cells. MATERIALS/METHODS: A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and protein expression analysis of osteoblastic genes were carried out to assess the osteoblastic proliferation and differentiation. RESULTS: The results indicated that treatment of SLE promoted the proliferation of MC3T3-E1 cells and improved ALP activity. And, SLE treatment significantly promoted mineralized nodule formation compared with control. In addition, cells treated with SLE significantly upregulated protein expression of ALP, type 1 collagen, bone morphogenetic protein 2, runt-related transcription factor 2, osterix, and osteoprotegerin. CONCLUSIONS: The results demonstrate that SLE promote differentiation inducement and proliferation of osteoblasts and, therefore may help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.28 no.3
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• pp.216-225
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• 2002

Hyaluronic acid (HA) is an almost essential component of extracellular matrices. Early in embryogenesis mesenchymal cells migrate, proliferate and differentiate, in part, because of the influence of HA. Since the features of embryogenesis are revisited during wound repair, including bone fracture repair, this study was initiated to evaluate whether HA has an effect on calcification and bone formation in an in vitro system of osteogenesis. Mouse calvaria Pre-osteoblast (MC3T3-E1) cells were cultured in ${\alpha}-MEM$ medium with microorganism-derivative hyaluronic acid that was produced by Strep. zooepidemicus which of molecular weight was 3 million units. The dosages were categorized in each 0.5, 1.0 and 2.0 mg/ml concentration experimental groups. After 2 and 4 days cultures in expeirmental and control groups, the tendency of cell proliferation, MTT assay, protein synthesis ability, collagen synthesis and alkaline phosphatase activity were analysed and bone nodule formation capacity were measured with Alizarin Red S stain after 29 days cultures. The cell proliferation was increased in time, especially the group of 0.5 and 1.0 mg/ml concentration of HA were showed prominent cell proliferation. After 2 and 4 days culture, experimental groups in general were greater cell activity in MTT assay. The protein synthesis was increased in all experimental groups compared to control group, especially most prominent in 1.0 mg/ml concentration group. The collagen synthesis capacity were increased in HA experimental groups, especially prominent in 1.0 mg/ml group and the activity of alkaline phosphatase were increased, especially also prominent in 1.0 mg/ml group, compared to control group. Above these, the activity of mouse carvarial pre-osteoblast cells was showed greater bone osteogenesis activity in all applied HA experimental group, especially group of 1.0 mg/ml concentration of HA.

• Journal of the korean academy of Pediatric Dentistry
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• v.27 no.1
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• pp.151-160
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• 2000

The effect of concentration as factor in cytotoxicity, protein synthesis and alkaline phosphatase activity was compared for pulpotomy medicaments (formaldehyde, formocresol, paraformaldehyde, ferric sulfate) Human pulp fibroblasts were exposed to a range of concentrations(0.01, 0.05, 0.10, 0.25, 0.50, $1.00{\mu}l/ml$) of each agents, for period of 24hrs. The cell activities were evaluated by MTT assay, protein assay and alkaline phosphatase activity examination. The results as follows : 1. After 24hrs culture, pulp fibroblasts adding formaldehyde, formocresol and paraformaldehyde were suppressed cell activities with concentration increasing, but, no depression of cell activities by ferric sulfate. No significant difference was in formaldehyde, formocresol and paraformaldehyde. 2. Protein synthesis by pulpotomy agents were not significant difference in pulp fibroblasts but protein synthesis were a little decreased by paraformaldehyde. 3. Alkaline phosphatase activity was a little decreased by pulpotomy medicaments.

• The korean journal of orthodontics
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• v.30 no.2 s.79
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• pp.205-214
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• 2000

Recently, the magnetic force has been considered as a method for a more efficient tooth movement. The purpose of this study was to evaluate the effects of different static magnetic fields of Nd-Fe-B magnet on MC3T3-E1 cells by measuring the alkaline phosphatase activity and observing the amount of stained alkaline phosphatase. For measuring of alkaline phosphatase activity, MC3T3-E1 cells were seeded in first and third row of 12 well culture plates. And Nd-Fe-B magnets were positioned under the first column of first and third row to apply different static magnetic fields(first column:100mT ; second column:4.6mT ; third column:0.5mT ; forth column:0.0mT) to the cells for 7, 13, 19, and 25 days. For staining of alkaline phosphatase, MC3T3-E1 cells were seeded in 100mm culture plates. And Nd-Fe-B magnets were positioned under the corner of plates to apply different static magnetic fields(magnet side:100mT : the opposite side:0.5mT) to the cells for 7, 13, 19, and 25 days. The results were as follows : 1. ALP activity was increased until day 19 in biochemical determination as well as in histochemical staining, 2. The application of higher magnetic field(100mT) suppressed ALP activity at day 13, 19, 25. On the contrary, the application of the lower magnetic field(4.6mT, 0.5mT) significantly enhanced the ALP activity. 3. Consistent with enzyme assay, histochemical staining of ALP also demonstrated that higher magnetic field(100mT) suppressed ALP activity, lower one(0.5mT) enhanced.

• Bulletin of the Korean Chemical Society
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• v.25 no.1
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• pp.115-118
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• 2004

We have examined the feasibility of using the specific interaction between mistletoe lectin I (ML I) and ${\beta}$-Dgalactose instead of the anti-ML I antibody in developing a homogeneous type competitive binding assay for ML I. We also have examined the feasibility of adapting the biotin/avidin mediated homogeneous assay for this system. Alkaline phosphatase (AKP) was employed as a single substrate enzyme label. The dose-response curve shows a detection range of 1-25 ${\mu}$g/mL and a linear response with a correlation coefficient of 0.99. To demonstrate the analytical utility of this method, 10 ${\mu}$g/mL of ML I was spiked into distilled water. The results show that the mean recovery was 10.03 ${\mu}$g/mL with an SD of 0.18. The difference between the spiked value and the mean recovery was 0.03 ${\mu}$g/mL, with a relative error of 0.3 and 1.6 % of RSD.

• Journal of Microbiology
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• v.35 no.2
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• pp.87-91
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• 1997

Monoclonal antibodies were prepared in order to an assay method for Vibrio vulnificus. Sixteen mouse ybridoma cell lines were established by immunization of whole cell antigen to BALB/c mice, fusion with SP2/O myeloma cells, and cloning. Most of them secreted IgM.lambda. antibodies. A sandwich enzyme-linked immunosorbent assay was developed with rabbit anti-V. vulnificus polyclonal antibodies as capture antibody, an IgM monoclonal antibody as detector antibody, and goat anti-mouse IgM-alkaline phosphatase conjugate as developer antibody. The range of detection was 10$\^$4/ to 10 V. vulnificus cells per microplate well. When four related Vibrio species were tested for cross-reactions, V. parahaemolyticus showed 3.5% reactively and V. carchariae, V. fluvialis, and V. furnisii showed negligibal (<1%) cross-reactivity.