• Title/Summary/Keyword: In vitro regeneration

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Hydroxylation of Compactin (ML-236B) by CYP105D7 (SAV_7469) from Streptomyces avermitilis

  • Yao, Qiuping;Ma, Li;Liu, Ling;Ikeda, Haruo;Fushinobu, Shinya;Li, Shengying;Xu, Lian-Hua
    • Journal of Microbiology and Biotechnology
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    • v.27 no.5
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    • pp.956-964
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    • 2017
  • Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steady-state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The $k_m$ and $k_{cat}$ values for compactin were $39.1{\pm}8.8{\mu}M$ and $1.12{\pm}0.09min^{-1}$, respectively. The $k_{cat}/K_m$ value for compactin ($0.029min^{-1}{\cdot}{\mu}M^{-1}$) was lower than that for diclofenac ($0.114min^{-1}{\cdot}{\mu}M^{-1}$). Spectroscopic analysis showed that CYP105D7 binds to compactin with a $K_d$ value of $17.5{\pm}3.6{\mu}M$. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

Response of Odontoblast to the Bio-Calcium Phosphate Cement

  • Kim, Jin-Woo;Kim, Sung-Won;Kim, Gyoo-Cheon;Kim, Yong-Deok;Kim, Cheol-Hun;Kim, Bok-Joo;Kim, Uk-Kyu
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.33 no.4
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    • pp.301-307
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    • 2011
  • Purpose: If the tooth structure is damaged, then it is impossible to regenerate the tooth. The materials used to restore the tooth structure are not related to the composition of the tooth. The materials used to restore the structure can't replace the natural tooth because they just fill the defective structure. Calcium phosphate cement remineralizes the dentin and almost replaces the natural tooth, but there are some disadvantages. We conducted basic tests with Biomimetic CPC (Bio-CPC) to make sure of the possibility of the biomaterial to remineralize the defective tooth structure. Methods: In this study, the bioactivity and biocompatibility of Bio-CPC were evaluated for its potential value as the bio-material for regeneration of damaged tooth structure by conducting a cell toxicity assay (WST-1 assay), a cytokinesis-block micronucleus assay, a chromosomal aberration test, total RNA extraction and RT-PCR on MDPC-23 mouse odontoblast-like cells. Results: The in vitro cytotoxicity test showed that the Bio-CPC was fairly cytocompatible for the MDPC-23 mouse odontoblast-like cells. Conclusion: Bio-CPC has a possibility to be a new biomaterial and further study of Bio-CPC is needed.

Enhanced Seed Development in the Progeny from the Interspecific Backcross (Fagopyrum esculentum ${\times}$ F. homotropicum) ${\times}$ F. esculentum

  • Shin, Dong-Hoon;Kamal, A H M;Yun, Young-Ho;Bae, Jeong-Sook;Lee, Yun-Sang;Lee, Moon-Soon;Chung, Keun-Yook;Woo, Sun-Hee
    • Korean Journal of Plant Resources
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    • v.22 no.3
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    • pp.209-214
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    • 2009
  • To facilitate the introgression of F. esculentum into the traits of F. homotropicum, several accessions of the hybrids between these two species were pollinated with F. esculentum as the recurrent parent. The embryo in vitro rescue was performed to increase the recovery of backcross progenies. The $F_{2}$ generation was more amenable than $F_{1}$ hybrids to produce backcross progenies. The $F_{1}$ hybrids were backcrossed twice with common buckwheat (pin-type F. esculentum) (recurrent backcrossing). Also, alternate backcrosses with common buckwheat and F. homotropicum (congruity backcrossing) were carried out. Pollen tube growth of BC$F_{1}$ ${\times}$ F. esculentum (thrum) and F. homotropicum ${\times}$ BC$F_{1}$ was the disturbed penetration exceeded for all initial interspecific hybrids, and its requirement was proportionally lower when the common buckwheat was used as the recurrent parent and as the last parent of congruity hybrids. Effects of both common buckwheat and F. homotropicum on seed success rate for hybridization were observed. Growth of hybrid embryos before rescue, regeneration of mature hybrids all increased recurrent and congruity backcrosses and inter-crosses between $F_{1}$ plants and selected fertile plants of the second congruity backcrosses.

Effects of biphasic calcium phosphate on bone formation in human fetal osteoblasts (Biphasic Calcium Phosphate가 태아골모세포의 골 형성에 미치는 영향)

  • Shin, Kye-Chul;Jang, Kil-Young;Lee, Myoung-Ku;Yoon, Ho-Sang;Song, Jae-Bong;Kim, Hyun-A;Pi, Sung-Hee;Shin, Hyung-Shik;You, Hyung-Keun
    • Journal of Periodontal and Implant Science
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    • v.35 no.1
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    • pp.77-85
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    • 2005
  • 목적 : 이 연구의 목적은 치과 영역에서 골 재생을 촉진하기 위해, 현재 많이 사용하고 있는 BDX(bovinederived xenograft)와 비교하여 BCP(biphasic calcium phosphate)의 효과를 알아보기 위함이다. 실험 재료 및 방법: 본 연구는 태아골모세포주(hFOB 1.19)를 사용하였으며, 사용된 골 이식재에 따라 2개의 실험군으로 구분하였고, 각 실험에 적절한 농도의 BDX와 BCP를 첨가하였다. 그리고, 세포 증식도 검사, 교원질 합성량 분석, 염기성 인산분해효소 활성도 측정, Western blot 분석을 통한 OC과 BSP의 발현 정도등의 실험을 진행하였다. 결과 : BDX와 BCP는 대조군과 비교하여 세포 증식에서 유의한 차이가 없었지만, 교원질 합성량, 염기성 인산분해효소의 활성, 그리고 OC과 BSP의 발현에 있어 대조군과 비교하여 유의하게 증가를 보였다. 그러나, 두 이식재간의 유의한 차이는 보이지 않았다. 결론 : 본 실험실적 연구에서 BCP는 골모세포분화에 긍정적인 영향을 미침으로써 효과적인 이식재로 사용할 수 있음을 가늠할수 있었다.

THE EFFECT OF GROWTH FACTORS ON OSTEOGENIC DIFFERENTIATION OF ADIPOSE TISSUE-DERIVED STROMAL CELLS (지방기질유래 줄기세포의 골 분화 시 성장인자의 효과)

  • Kim, Uk-Kyu;Choi, Yeon-Sik;Jung, Jin-Sup
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.32 no.4
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    • pp.327-333
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    • 2006
  • Future cell-based therapies such as tissue engineering will benefit from a source of autogenous pluripotent stem cells. There are embryonic stem cells (ESC) and autologous adult stem cells, two general types of stem cells potentilally useful for these applications. But practical use of ESC is limited due to potential problems of cell regulation and ethical considerations. To get bone marrow stem cells is relatively burden to patients because of pain, anesthesia requirement. The ideal stem cells are required of such as the following advantages: easy to obtain, minimal patient discomfort and a capability of yielding enough cell numbers. Adipose autologus tissue taken from intraoral fatty pad or abdomen may represent such a source. Our study designed to demonstrate the ability of human adipose tissue-derived stromal cells (hATSC) from human abdominal adipose tissue diffentiating into osteocyte and adipocyte under culture in vitro conditions. As a result of experiment, we identified stromal cell derived adipose tissue has the multilineage potentiality under appropriate culture conditions. And the adipose stromal cells expressed several mesenchymal stem cell related antigen (CD29, CD44) reactions. Secondary, we compared the culture results of a group of hATSC stimulated with TGF-${\beta}$1, bFGF with a hATSC group without growth factors to confirm whether cytokines have a important role of the proliferation in osteogenic differentiation. The role of cytokines such as TGF-${\beta}$1, bFGF increased hATSC's osteogenic differentiation especially when TGF-${\beta}$1 and bFGF were used together. These results suggest that adipose stromal cells with growth factors could be efficiently available for cell-based bone regeneration.

Effect of Propofol Preconditioning on Hypoxic-Cultured Human Osteoblast

  • Yoon, Ji Uk;Shin, Sang Wook;Park, Bong Soo;Kim, Yong Ho;Woo, Mi Na;Yoon, Ji Young;Kim, Cheul Hong
    • Journal of The Korean Dental Society of Anesthesiology
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    • v.14 no.2
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    • pp.107-114
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    • 2014
  • Background: Angiogenesis has been recognized an essential precondition for osteogenesis. Because reduction and disruption of the blood supply to tissue cause tissue hypoxia, pathological bone loss affected by hypoxia often can occur in various clinical conditions. The effects of propofol on the process of osteogenesis have received little direct attention. Therefore, we investigated the effect of propofol on the growth and function of osteoblasts under hypoxic condition. Methods: After propofol (3, 30, $300{\mu}M$) preconditioning for 2 hours, hFOB 1.19 human osteoblast cells were cultured under 1 % oxygen tension for 48 hours. Using real time PCR and western blot analysis, we analyzed the expression of, BMP-2, TGF-${\beta}1$, type I collagen, osteocalcin, HIF-1s and Akt. Cell viability was also determined by MTT assay. Results: Propofol preconditioning on hypoxic-cultured osteoblast promoted the expressions of BMP-2, TGF-${\beta}1$, type I collagen and osteocalcin and induced hypoxia-mediated HIF-1 activation and the expression of Akt protein. Propofol with $300{\mu}M$ significant decreased cell viability compared to control. Conclusions: Clinically relevant concentrations of propofol are not cytotoxic to hypoxic osteoblasts in vitro. Propofol preconditioning on hypoxic-cultured osteoblast stimulates proliferation and differentiation of osteoblast through induced expression of BMP-2, TGF-${\beta}1$, type I collagen and osteocalcin. Propofol might promote angiogenesis and bone regeneration under hypoxic condition.

In Vitro Plant Regeneration for Mass Propagation of Epimedium koreanum Nakai (삼지구엽초의 다량번식을 위한 기내 식물체 분화)

  • Han, Young-Hee;Choi, Byoung-Ryourl;Soh, Ho-Seob;Lee, Seong-Jae;Choi, Young-Jin;Kim, Se-Young
    • Horticultural Science & Technology
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    • v.18 no.6
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    • pp.834-838
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    • 2000
  • As an endeavor to establish a micropropagation system for Epimedium koreanum Nakai., this study was carried out to define methods to disinfect its explants and media for callus induction, proliferation and plant regeneration. The lowest infection rates by fungi or bacteria on apical and axillary bud explants of rhizome were observed when they were immerged in 0.3% NaOCl solution for 20 min after soaked in 0.1% $AgNO_3$ solution for 30 min, but leaf explants were seldom infected with fungi or bacteria by this disinfectant method. The highest rate of plantlet formation was obtained from the explants disinfected in 0.3% NaOCl solution for 20 min after soaked in 0.1% $AgNO_3$ solution for 60 min for tip buds and in 0.1 % $AgNO_3$ solution for 30 min for axillary buds of rhizome. Induction rate of callus was the highest from the explants disinfectd in 0.3% NaOCl solution for 20 min after soaked in 0.2% $AgNO_3$ solution for 15 min. Callus growth was proper in a modified 1/2 MS medium including half strength of $NH_4NO_3$ with $0.02-0.2mg{\cdot}L^{-1}$ BA and $2.0mg{\cdot}L^{-1}$ NAA. Low rate of plantlet regeneration was obtained in 1/2 UM or 1/2 White medium with $2.0mg{\cdot}L^{-1}$ BA and $0.2mg{\cdot}L^{-1}$ AA.

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Design and Fabrication of Nasal-Implant-Shaped Scaffold and Regeneration of Nasal Cartilage Tissue for Rhinoplasty (코 성형을 위한 코 보형물 형태의 인공지지체 설계 및 제작과 코 연골조직의 재생)

  • Jung, Jin-Woo;Jang, Jin-Ah;Shim, Jin-Hyung;Kim, Sung-Won;Cho, Dong-Woo
    • Transactions of the Korean Society of Mechanical Engineers B
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    • v.36 no.11
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    • pp.1111-1117
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    • 2012
  • Implants for rhinoplasty should ideally be biocompatible and possess long-term stability after implantation. Silicone implants are most widely used for rhinoplasty. However, these implants suffer from problems related to high extrusion and infection rates. To minimize these complications, we propose a novel augmentation rhinoplasty technique using tissue engineering. To demonstrate its feasibility, a nasal-implant-shaped scaffold was designed using commercialized CAD software and fabricated using a Multi-head Deposition System, which is a solid freeform fabrication system that dispenses material. In vitro cell proliferation and chondrogenic differentiation tests were carried out using nasal septal chondrocytes.

Gene Transformation of Ailanthus altissima Swingle by Agrobacterium tumefaciens (외래유전자(外來遺傳子)에 의(依)한 가중나무의 형질전환(形質轉換))

  • Park, Young Goo;Huh, Kyung;Choi, Myung Suk
    • Current Research on Agriculture and Life Sciences
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    • v.10
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    • pp.137-145
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    • 1992
  • An efficient transformation system was established for Ailanthus altissima utilizing the binary system of A. tumefaciens strain LBA4404. Callus was initiated from small portions of cambium tissue of A. altissima in vitro. Optimum regeneration was achieved with Murashige and Skoog(MS) medium containing 0.01mg/${\ell}$ 2, 4-D, 0.5mg/${\ell}$ BAP, 3%(w/v) sucrose and 0.75% agar. The multiplication of explants remarkably showed up on medium containing 1.0mg/${\ell}$ BAP. Leaf discs or internodal stem segments were inoculated with A. tumefaciens strain LBA 4404 containing the binary vector pPMB 101, which has both ${\beta}$-glucuronidase (GUS) marker gene and neomycin phosphotransferase II (NPT II) gene. Shoots had been regenerated from 24 lines out of inoculative 50 lines. Transformants were selected by their ability to grow on medium containing kanamycin sulphate (100mg/${\ell}$). Putative transformation was confirmed by GUS assays. Five GUS-positive plantlets were obtained which confirmed that this marker gene has been transferred into A. altissima.

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The Effect of Chlorhexidine on the formation of bone nodules by Periodontal ligament Cells in Vitro (사람치주인대섬유모세포에 의한 골결절 형성시 Chlorhexidine의 효과)

  • Choi, Hui-Jun;Ji, Suk;Kook, Joong-Ki;Jang, Hyun-Seon;Park, Joo-Cheol;Kim, Heung-Joong;Kim, Chong-Gwan;Kim, Byung-Ock
    • Journal of Periodontal and Implant Science
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    • v.36 no.2
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    • pp.375-383
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    • 2006
  • 사람치주인대섬유모세포(human periodontal ligament fibroblast, PDLF)의 기능 손상과 클로르헥시딘(Chlorhexidine, CHX)의 세포독성에 관한 분자적인 기전은 최근까지도 불명확하다. 이 연구의 목적은 PDLF에 의한 골결절 형성에 있어서 CHX의 효과를 평가하고, 치주수술후에 치주병원균의 최소억제농도(minimal inhibitory concentration, MIC)를 평가하고자 하였다. CHX의 세포독성을 평가하기 위해서 MTT assay법을 실시하였다. CHX은 0.12%에서 0.00012%까지, 즉 10-1000배로 희석시킨 후 30, 60, 120초 동안 PDLF에 적용되었고, 석회화된 결절은 alizarin red 용엑에 염색되었다. 치주병원균에 대한 CHX의 MIC가 평가되었다. 이 연구 결과, 세포생존율 검사에서는, 단지 0.12% CHX 에 노출되었던 세포들만 세포 증식 소견을 다소 나타내었다. 모든 CHX 농도(0.12%-0.00012%)에서 PDLF에 의한 골결절 형성은 의미있는 감소를 나타내었다. 또한 치주병원균에 대한 CHX의 MIC는 0.0012%로 나타났다. PDLF의 골결절 형성에 영향을 주는 농도(0.00012%)는 세포독성을 나타내는 농도(0.12%)보다 더 낮은 농도를 보였고, 치주병원균의 최소억제에 필요한 농도는 0.0012%로 나타났다. 이런한 결과들은 통상적으로 상용되는CHX이 PDLF에 의한 골결절 형성에 있어서 영향을 미칠 수 있음을 시사하였다.