• Food Science and Biotechnology
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• 제17권2호
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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.

• 한국토양동물학회지
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• 제5권1호
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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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• 제25권3호
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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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• 제35권6호
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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.

• 한국식품영양학회지
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• 제21권1호
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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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• 제30권7호
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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.

• 미생물학회지
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• 제52권1호
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• pp.10-17
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• 2016

항체의 특이적 결합을 분석하는 효소면역분석법은 항원의 탐지를 위해 주로 horseradish peroxidase (HRP) 또는 alkaline phosphatase (AP) 등의 효소를 사용한다. 이때 효소를 주로 화학적으로 항체에 결합시켜 사용하게 되는데 이 과정이 복잡하며 불규칙하게 일어나서 항체 및 효소의 기능을 감소시키게 된다. 또한 대부분의 효소면역분석법에서는 주로 일차 항체의 항원결합을 탐지하기위해 이차 항체를 사용하는데, 즉 이차 항체에 결합한 효소의 기질발색에 의해 일차 항체의 항원결합을 탐지하므로 이차 항체가 요구 되어질 뿐만 아니라 이차 항체의 일차 항체에 대한 반응을 위한 부가적인 배양시간이 필요하다. 더욱 더 중요한 것은 이차 항체만의 비특이적 항원 결합 역시 제거되어져야 한다. 본 연구에서는 대장균의 genomic DNA로부터 PCR을 통해 alkaline phosphatase 유전자(Sadeghi et al., 2008)를 증폭 분리한 다음 이를 TRAIL (tumor necrosis factor ${\alpha}$ related apoptosis induced ligand) receptor인 death receptor 4 (DR4)에 특이적으로 결합하는 hAY4 single-chain Fv (ScFv)에 융합시킨 재조합 ScFv-AP 형태로 대장균에서 발현시켜 정제하였다. 정제된 hAY4 ScFv-AP는 SDS-PAGE에서 단량체(monomer) 분자량인 73.8 kDa을 나타내었다. 그러나 size-exclusion chromatography(SEC)에서는 147.6 kDa을 나타내는 결과를 통해 hAY4 ScFv-AP는 AP의 자연적인 비공유결합에 의해 이량체(dimeric form)형성이 유도되어짐을 확인하였다. 또한 ELISA, Western blot 그리고 immunocytochemistry에서 이차 항체 없이 일차 항체 hAY4 ScFv에 직접 융합된 AP의 기질발색에 의해 ScFv 일차 항체의 특이적 항원결합을 나타내었다. 요약하면 hAY4 ScFv와 대장균의 alkaline phosphatase 유전자를 융합시켜 대장균에서 수용성 형태로 성공적으로 정제하였으며 정제된 ScFv-AP 융합단백질은 ELISA, Western blot 및 immunocytochemistry에서 항원결합력을 나타냈으며 또한 구매에 따른 고비용, 부가적인 배양시간 및 비특이적 결합에 의한 오류 등의 문제점을 갖는 이차 항체를 사용하지 않고 직접적인 항원결합력을 나타내었다.

• Journal of Nutrition and Health
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• 제31권3호
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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.

• 대한소아치과학회지
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• 제28권2호
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• pp.238-246
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• 2001

본 실험은 탁월한 골유도능으로 관심의 대상이 되고 있는 BMP의 세포내 신호 전달자로 알려진 Smad 1과 Smad 5가 조골세포 초기 분화표지인자인 ALP 유전자의 발현에 미치는 영향 및 그 조절기전을 알아보고자 하였다. BMP 처리 없이도 Smad에 의해 ALP가 발현되는가를 알아보기 위해 Smad 1과 Smad 5가 각각 stably transfection된 C2C12 세포를 3일간 배양후 histochemical assay를 하였고, Smad 1과 Smad 5의 expression vector와 ALP promoter vector를 transient co-transfection한 후 ALP promoter activity를 측정하였다. Smad에 의한 BMP의 효과를 알아보기 위해서 100ng/ml의 BMP-2를 처리한 군과 처리하지 않은 군으로 나누어 세포를 배양한후 ALP 유전자의 발현을 northern blot analysis로 확인 하였다. Smad가 ALP 유전자의 발현을 직접적으로 조절하는가를 알아보기 위해서는 단백질 합성억제제인 cycloheximide를 전처리하여 ALP 유전자의 발현을 northern blot analysis하였다. 이상의 실험결과 다음과 같은 결론을 얻었다. $\cdot$ Smad 1과 Smad 5가 과발현된 세포에서는 BMP 처리없이도 ALP가 발현된다. $\cdot$ Smad 1과 Smad 5가 과발현된 세포에서 BMP 처리후 ALP 발현 증가율이 대조군 보다 현저히 높게 나타나 Smad가 BMP 효과를 증가시킨다는 것을 알 수 있다. $\cdot$ Smad는 새로운 단백질의 합성을 통해 ALP 유전자를 발현시킨다.

• 대한치과보철학회지
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• 제49권4호
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• pp.333-340
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• 2011

연구 목적: 이 연구의 목적은 수산화인회석 코팅 결정도가 조골세포의 분화에 미치는 영향을 조사하기 위함이다. 연구 재료 및 방법: 제작된 모든 시편은 양극산화과정을 거치면서 티타늄 표면에서 산화막을 형성하여 표면 거칠기를 증가시켰고 각 시편의 표면을 IBAD (ion beam-assisted deposition) 시스템을 이용하여 HA (hydroxyapatite) 코팅하였다. HA의 코팅이 완료된 시편들은 전기가열로(AJ-SB3, AJEON Heating Industrial Co., Ltd, Seoul, Korea)에 넣어 각 실험군별로 $100^{\circ}C$, $300^{\circ}C$, $500^{\circ}C$, $800^{\circ}C$까지 온도를 상승시켜 열처리하였다. HA 코팅을 실시하지 않은 군은 대조군으로 설정하고(control) 소결된 각각의 그룹은 HA100, HA300, HA500, HA800으로 구분하여 설정하였다. 시편 표면의 물리적 성질은 표면 거칠기 테스트, XRD, SEM으로 평가되었다. 수산화인회석 코팅의 결정도의 효과는 조골세포의 분화에 의해 연구되었는데 1, 3, 5, 7일 후에 평가되었다. 성장과 분화 역학은 세포증식능평가, ALP (alkaline phosphatase) 활성능 평가에 의해 조사되었다. 결과: 표면 거칠기는 양극산화 처리 후 IBAD 방식으로HA를 코팅하여도 그 거칠기에는 별 다른 차이가 없음을 보였다. X선 회절분석 결과 $100^{\circ}C$와 $300^{\circ}C$에서 소결한 시편은 HA의 결정화가 없는 무정형상태이며 $500^{\circ}C$와 $800^{\circ}C$에서 소결한 시편의 HA에서는 결정화 상태가 나타났다. 표면에 배양된 조골 세포의 증식능을 측정한 결과 1일과 3일에서는 각 실험군간의 유의할만한 차이가 있었으나, 5일과 7일에는 각 대조군과 실험군 모두 유의성 있는 차이를 보이지 않았다. ALP 활성능은 HA100과 HA300보다 HA500과 HA800이 더 높았다. 결론: 본 연구의 결과에서 양극산화처리된 티타늄표면에 이온빔보조증착법을 이용하여 수산화인회석을 코팅 후 소결할 때 $500^{\circ}C$의 소결온도가 수산화인회석코팅층의 결정화와 HOS (human osteosarcoma cells) 세포의 증식과 분화에 효과가 좋은 것으로 나타났다.