• Food Science and Biotechnology
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• v.17 no.2
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• pp.434-437
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• 2008

The aim of this study was to investigate whether Monascus-fermented soybean extracts (MFSE) containing natural estrogen-like compounds such as isoflavones and mevinolins has potential effects on human osteoblast-like SaOS2 cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and alkaline phophatase (ALP) assaies. MFSE exerted biphasic dose-dependent effect; stimulating osteoblastic activity at low concentrations and inhibiting SaOS2 cells viability at high concentrations. At $10^{-8}-10^{-4}\;mg/mL$, MFSE is not only non-cytotoxic but also induced comparatively high ALP activity on SaOS2 cells. ALP activity (%) significantly increased (220.1%, p<0.05) when SaOS2 cells were treated with MFSE at a concentration of $10^{-5}\;mg/mL$, whereas slowly increased (185.6%, p<0.05) in unfermented soybean extracts (UFSE) at $10^{-3}\;mg/mL$. The potentially greater ALP activity of MFSE compared to the UFSE might partially be caused by its mevinolin, which was derived from the soybean during Monascus-fermentation. Our findings indicate that supplementation of MFSE may accelerate the speed of intracellular ALP synthesis by the bone cells when provided at optimal dosages.

• The Korean Journal of Soil Zoology
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• v.5 no.1
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• pp.39-46
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• 2000

The distribution and some properties of alkaline phosphatase (ALP) were investigated in the midgut of the earthworm, Eisenia andrei. The ALP activity appeared to be highly polarized toward the luminal side of epithelium, with minor ALP activity in chloragogue tissue. The epithelial and chloragogeneous tissues contained approximately 85 and 15% of total intestinal ALP activity, respectively. The optimal temperature was approximately 37$^{circ}C$ and isoelectricpoint was estimated to be 4. The treatment of neuraminidase and PtdIns-PLC failed to change the migration rate of ALPs. Also, these ALPs appeared to have a wide range of substrate specificity. The relationship between the properties and physiological significances of the midgut ALPs in Eisenia andrei was discussed.

• KSBB Journal
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• v.25 no.3
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• pp.257-264
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• 2010

Bone morphogenetic protein-7 (BMP-7) is one of important growth factors for skeletal development and bone growth. In this work, BMP-7 was efficiently expressed in recombinant Bacillus subtilis. The mature BMP-7 protein indicated molecular weight of 15.4 kDa by Western blot assay and was secreted into culture medium with 0.35 ng/mL. The extracellular and intracellular rhBMP-7 proteins were purified by using a FPLC system with an ion exchange column and a gel filtration column. The extracellular and intracellular rhBMP-7 proteins had finally a 57.1% purity and a 36.2% purity, respectively. The purified rhBMP-7 proteins showed an intact biological activity which stimulated alkaline phophatase (ALP) activity in MC3T3-E1 cells.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.35 no.6
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• pp.397-402
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• 2009

The purpose of this study is to investigate the effects of alendronate and pamidronate on proliferation and the alkaline phosphatase activity of human bone marrow derived mesenchymal stem cells and to relate the results with bisphosphonate related osteonecrosis of the jaw(BRONJ). With the consent of patients with no systemic disease and undergoing iliac bone graft, cancellous bone was collected to obtain human bone marrow derived mesenchymal stem cells through cell culture. 96 well plate were prepared with a concentration of $10^4$cell/ well. Alendronate and pamidronate were added to each well with the concentration of $10^{-6}M$, $10^{-8}M$ and $10^{-10}M$, respectively. Then proliferation capacity of each well was evaluated with the cell counting kit. 24 well plates were prepared with a concentration of $10^5$cell/ml/well and with the bone supplement, alendronate and pamidronate were added with the concentration of $10^{-6}M$, $10^{-8}M$ and $10^{-10}M$, respectively on each plate. The plates were cultured for either 24 or 72 hours. Then the cells were sonicated to measure the alkaline phosphatase activity and protein assay was done to standardize the data for analysis. As the concentration of alendronate or pamidronate added to the culture increased, the proliferation capacity of the cells decreased. However, no statistical significance was found between the group with $10^{-10}M$ of bisphophonate and the control group. Pamidronate was not capable of increasing the alkaline phosphatase activity in all trials. However, alkaline phosphatase activity increased with 24 hours of $10^{-8}M$ of alendronate treatment and with 48 hours of $10^{-10}M$ of alendronate treatment. Cell toxicity increased as the bisphosphonate concentration increased. This seems to be associated with the long half life of bisphosphonate, resulting in high concentration of bisphosphonate in the jaw and thus displaying delayed healing after surgical procedures. Alendronate has shown to increase the alkaline phophatase activity of human bone marrow derived mesenchymal stem cells. However, this data is insufficient to conclude that alendronate facilitates the differentiation of human bone marrow derived mesenchymal stem cells. Further studies on DNA level and animal studies are required to support these results.

• The Korean Journal of Food And Nutrition
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• v.21 no.1
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• pp.71-78
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• 2008

This study examined the effects of mulberry-leaf powder Tofu(MPT) on the anthropometric measurements, bone mineral density(BMD), biochemical markers of bone turnover, serum lipid levels of and macrominerals status in 30 female university students living in Choongnam with low dietary Ca intake was low(mean daily $intake=524.66{\pm}21.47$ mg/day). The subjects received the MPT supplemented(100 g/day) for 4 weeks. The nutrient content per 100 g MPT was 84.10 kcal(energy), 8.98 g(protein), 0.53 mg(fiber), 211.33 mg(Ca) and 1.59 g(fat). Anthropometric measurements, 24-hour dietary recall, carpus BMD using DEXA, select BMD biomarckers(serum alkaline phophatase activity, osteocalcin, urinary deoxypyridinoline), serum protein levels, albumin, and glucose levels, lipid profiles(cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol) and serum macrominerals(Ca, Ca/P ratio, Mg) were analyzed before and after MPT supplementation. Following MPT supplementation, there was no significant differences in anthropometric measurements, nutrient intakes, carpus BMD, serum alkaline phosphatase activity, osteocalcin, and urinary deoxypyridinoline, serum levels of protein, albumin, and glucose or lipid profiles with AI, HTR, LHR and CRF. However, there were significant increases in dietary calcium as well as decreases in serum triglycerides with MPT supplementation. Although further research is needed, these results that regular intake MPT could prevent bone and cardiovascular diseases.

• Journal of Nutrition and Health
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• v.30 no.7
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• pp.813-824
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• 1997

This study was designed to investigate the effect of dietary calcium and phosphate levels on calcium and bone metabolism in rats. The rats were divided into six groups and each of the groups was fed diets with different Ca/P ratios. The experimental periods were 5 weeks . There was no significant different difference in dietary intake, body weight gain, and organ weight among the groups with different calcium and phosphate intake levels. Fecal calcium excretion was not significantly different among the groups, but urinary calcium excretion was increased by the increase in Ca/P ratio. Fecal phosphate excretion was not different but urinary phosphate excretion was increased by the increase in dietary phosphate intake. There was no significant difference in serum alkaline phophatase activity and urinary hydroxyproline levels were not significantly different among the groups. The low calcium-high phosphate(0.25Ca-1.2% P) group showed the lowest total calcium content in femur and scapula. This may be due to it having the lowest Ca/P ratio among groups. The low calcium-high phosphate(0.2%Ca-1.2%P) group showed that mandible is almost lost and osteolyzed Harversian canal was expanded in femur. Results suggest that phosphate intake affects calcium and bone metabolism more with inadequate calcium nutrition that with adequate calcium intake. Thus , for normal bone growth and metabolism , adequate calcium intake and/or high Ca/P ratio are important.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.10-17
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• 2016

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

• Journal of Nutrition and Health
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• v.31 no.3
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• pp.279-288
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• 1998

This study explored the effect of calcium levels and/or ovariectomy on bone metabolism using female Sprague-Dawley weanling rats as a model . Rats received a low (0.1%) calcium diet for 8 weeks. The rats were then divided into three subgroups that were fed 0.1% ,0.5% and 1.5% calcium diets for 8 weeks after operation. The results of this experiment indicate that body weight gin was higher in ovariectomy groups than in sham groups regardless of calcium level and food intake. Serum Ca and P concentrations were of normal level regardless of calcium level and ovariectomy. Estrogen concentration was low in the ovariectomized group. Serum alkaline phophatase activity and urinary hydroxyproline have been used as markers of bone formation and resorption. These values were increased in ovariectomized groups. The weight, length and breaking force of femur were not significantly different between the groups. Ash, Ca, P and total lipid contents in femur and lumbar were decreased in the groups fed low calcium . Mg content was decreased in the ovariectomy and total protein content was not affected by calcium level and ovariectomy. The effect groups of ovarectomy on calcium contents of bone was more prominent in lumbar than in femur. In conclusion, though low calcium intakes during growth period may retard the attainment of peak bone mass, calcium supplementation after this period increased bone growth and mineral contents, but not significant effect in three calcium levels. Furthermore, calcium intake was shown to have a greater influence on the mineral contents of femur than of lumbar, and removal of endogenous estrogen production by ovariectomy was shown to be more deleterious on the ash and calcium contents of the lumbar than of femur.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.2
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• pp.238-246
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• 2001

Bone morphogenetic proteins(BMPs), members of the transforming growth factor $\beta$(TGF-$\beta$) superfamily were first identified as the factors that induce ectopic bone formation in vivo, when implanted into muscular tissue. Especially BMP-2 inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. In the molecular mechanism of the signal transduction of TGF-$\beta$ and related factors, intracellular signaling proteins were identified as Smad. In previous study, it has been reported that Smad 1 and Smad 5, which belong to the R-Smad family mediate BMP signaling, were involved in the induction of osteoblast differentiation in C2C12 cells. To understnad the role of Smads involved in osteogenic transdifferentiation in C2C12 cell, in present study, after we stably transfected C2C12 cells with each. Smad(Smad 1,Smad 5) expression vector, cultured for 3 days and stained for alkaline phophatase activity. ALP activity positive cells appeared in the Smad 1, Smad 5 stably transfected cell even in the abscence of BMP. After transiently co-transfected C2C12 cells with each Smad expression vector and ALP promoter, it was examined that Smad 1 and Smad 5 expression vector had increased about 2 fold ALP promoter activity in the abscence of BMP. These result suggested that both Smad 1 and Smad 5 were involved in the intracellular BMP signals which induce osteoblast differentiation in C2C12 cells. The effect of BMP on C2C12 cells with Smad 1, Smad 5 transfected were studied by using northern blot analysis. the treatment of BMP upregulated ALP mRNA level in three groups, especially upregulation of ALP was larger in Smad 1, Smad 5 transfected cell than control group. Pretreatment with cycloheximide($10{\mu}g/ml$), a protein synthesis inhibitor resulted in blocking the ALP gene expression even in BMP(100ng/ml) treated cell. These results suggested that Smad increased the level of ALP mRNA via the synthesis of a certain transcriptional regulatory protein.

• The Journal of Korean Academy of Prosthodontics
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• v.49 no.4
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• pp.333-340
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• 2011

Purpose: The aim of this study was to study the effect of hydroxyapatite (HA) coating crystallinity on the proliferation and differentiation of human osteosarcoma cells. Materials and methods: Surface roughness of the titanium disks increased by anodizing treatment and then HA was coated using ion beam-assisted deposition (IBAD). HA coating was crystallized by heat-treated at different temperature ($100^{\circ}C$, $300^{\circ}C$, $500^{\circ}C$, $800^{\circ}C$). According to the temperature, disks were divided into four groups (HA100, HA300, HA500, HA800). With the temperature, crystallinity of the HA coating was different. Anodized disks were used as control group. The physical properties of the disk surface were evaluated by surface roughness tests, XRD tests and SEM. The effect of the crystallinity of HA coating on HOS cells was studied in proliferation and differentiation. HOS cells were cultured on the disks and evaluated after 1, 3, 5, and 7 days. Growth and differentiation kinetics were subsequently investigated by evaluating cell proliferation and alkaline phosphatase activity. Results: Regardless of the heat-treated temperature, there is no difference on the surface roughness. Crystallinity of the HA was appeared in the groups of HA500, HA800. HOS cells proliferation, ALP activity were higher in HA500 and HA800 group than HA100 and HA300. Conclusion: Within the results of this limited study, heat treatment at $500^{\circ}C$ of HA coating produced by IBAD has shown greater effect on proliferation and differentiation of HOS cells. It is considered that further in vivo study will be necessary.