• Title/Summary/Keyword: ex vitro

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NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1α expression levels during hypoxia/reoxygenation injury

  • Vu, Thi Thu;Kim, Hyoung Kyu;Le, Thanh Long;Nyamaa, Bayalagmaa;Song, In-Sung;To, Thanh Thuy;Nguyen, Quang Huy;Marquez, Jubert;Kim, Soon Ha;Kim, Nari;Ko, Kyung Soo;Rhee, Byoung Doo;Han, Jin
    • The Korean Journal of Physiology and Pharmacology
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    • v.20 no.2
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    • pp.201-211
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    • 2016
  • Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment ($10{\mu}M$) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-$1{\alpha}$ ($PGC1{\alpha}$) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving $PGC1{\alpha}$ during cardiac HR injuries.

The Study of MHC class I Restricted CD8+ T Cell Mediated Immune Responses against Mycobacterium tuberculosis Infection: Evidence of M. tuberculosis S pecific CD8+ T Cells in TB Patients and PPD+ Healthy Individuals (MHC class I 분자들에 의해 제시되는 Epitope을 인지하는 CD8+ T 림프구의 결핵균 감염에 대한 면역반응의 연구: 결핵 환자와 PPD+ 건강개체에 존재하는 결핵균 항원에 특정한 CD8+ T세포)

  • Cho, Jang-Eun;Lee, Kyung Wha;Park, Seung Kyu;Cheon, Seon-Hee;Cho, Sang-Nae;Cho, Sungae
    • IMMUNE NETWORK
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    • v.3 no.3
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    • pp.235-241
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    • 2003
  • Background: The protective immunity against tuberculosis (TB) involves both CD4+ T cells and CD8+ T cells. In our previous study, we defined four Mycobacterium tuberculosis derived peptide epitopes specific for HLA-$A^*0201$ restricted CD8+ T cells ($ThyA_{30-38}$, $RpoB_{127-135}$, $85B_{15-23}$, $PstA1_{75-83}$). In this study, we investigated the immune responses induced by these peptide specific CD8+ T cells in latently and chronically infected people with TB. Methods: We characterized these peptide specific CD8+ T cell population present in PBMC of both TB patients and PPD+healthy people using IFN-${\gamma}$elispot assay, intracellular staining and HLA-A2 dimer staining. Results: The frequency of peptide specific CD8+ T cell was in the range of 1 to 25 in $1.7{\times}10^5$ PBMC based on ex vivo IFN-${\gamma}$ elispot assay, demonstrating that these peptide specific CD8+ T cell responses are induced in both TB patients and PPD+ people. Short term cell lines (STCL) specific for these peptides proliferated in vitro and secreted IFN-${\gamma}$ upon antigenic stimulation in PPD+ donors. Lastly, HLA-$A^*0201$ dimer assays indicated that $PstA1_{75-83}$ specific CD8+ T cell population in PPD+ healthy donors is heterogeneous since approximately 25~33% of $PstA1_{75-83}$ specific CD8+ T cell population in PPD+ healthy donors produced IFN-${\gamma}$ upon peptide stimulation. Conclusion: Our results suggest that MHC class I restricted CD8+ T cell mediated immune responses to M. tuberculosis infection are induced in both TB patients and PPD + people; however, the CD8+ T cell population is functionally heterogeneous.

Effect of combinatorial bone morphogenetic protein 2 and bone morphogenetic protein 7 gene delivery on osteoblastic differentiation

  • Bae, Young;Kim, Kyoung-Hwa;Kim, Su-Hwan;Lee, Chul-Woo;Koo, Ki-Tae;Kim, Tae-Il;Seol, Yang-Jo;Ku, Young;Rhyu, In-Chul;Chung, Chong-Pyoung;Lee, Yong-Moo
    • Journal of Periodontal and Implant Science
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    • v.39 no.sup2
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    • pp.279-286
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    • 2009
  • Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.

Large-scale Culture of Plant Cell and Tissue by Bioreactor System

  • Son, Sung-Ho;Park, Sung-Mee;Park, Seung -Yun;Kwon, Oh-Woung;Lee, Yun-Hee;Paek, Kee-Yoeup
    • Journal of Plant Biotechnology
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    • v.1 no.1
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    • pp.1-7
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    • 1999
  • Large-scale cultures of plant cell, tissue, and organ have been achieved by using BTBB. When different sized BTBBs (5 L, 20 L, 100 L, 300 L, and 500 L) were tested for the culture of yew cells (Taxus cuspidata Sieb. et Zucc.), cell growth increment reached to 94.5% in SCV after 24 days of culture with 30% of inoculation cell density. However, there were some variations in the production of taxol and its derivatives among the BTBBs of different size. Approximate 4 ㎎/l of taxol and 84 ㎎/l of total taxanes were obtained by using a 500L BTBB after 6 weeks of culture. With a 20L BTBB, about 20,000 cuttings of virus-free potatoes (cv. Dejima) could be obtained by inoculating 128 explants and maintaining 8 weeks under 16 hr light illumination. The frequency of ex vitro rooting of the cuttings revealed as more than 99% under 30% shade. By incorporating two-stage culture process consisting of multiple bulblet formation in solid medium and bulblet development in liquid medium, mass propagation of lily through bioreactor seemed to be possible. In the case of 'Marcopolo', the growth of mini-bulblets in BTBB was nearly 10 folds faster than that of the solid medium. Time course study revealed that maximum MAR yield of ginseng (Panax ginseng C. A. Meyer) in a 5 L and 20 L BTBB after 8 weeks of culture was 500 g and 2.2 ㎏, respectively. By cutting the MAR once and/or twice during the culture, the yield of root biomass could be increased more than 50% in fresh weight at the time of harvest. With initial inoculum of 500 g of sliced MAR in a 500 L BTBB, 74.8 ㎏ of adventitious root mass was obtained after 8 weeks of culture. The average content of total ginseng saponin obtained from small-scale and/or pilotscale BTBBs was approximately 1% per gram dry weight. Based on our results, we suggest that large-scale cultures of plant cell, tissue, and organ using BTBB system should be quite a feasible approach when compared with conventional method of tissue culture.

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Effect of explant's position and culture method on shoot proliferation and micro-cuttings for a rare and endangered species, Abeliophyllum distichum Nakai (희귀 및 멸종위기 식물 미선나무(Abeliophyllum distichum Nakai)의 절편위치 및 치상방법에 따른 기내증식 및 미세삽목)

  • Lee, Na Nyum;Kim, Ji-Ah;Kim, Yong-Wook;Choi, Yong Eui;Moon, Heung Kyu
    • Journal of Plant Biotechnology
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    • v.42 no.3
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    • pp.228-234
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    • 2015
  • Using either the apical or axillary bud of the endangered species Abeliophyllum distichum Nakai, we tested the effect of bud position and culture method on shoot proliferation and rooting. In shoot proliferation, the axillary bud explant was more effective than the apical bud and the effect was fostered by BA treatment, whereas no differences were observed in shoot elongation by the explant position. Spontaneous rooting was observed in the MS basal medium and resulted in conspicuous differences in the explant position : more than 80% in apical bud explant and 28% in axillary bud explant was achieved, respectively. The positional effects were also observed in BA pre-treatments: generally vertical culture method appeared to be better in shoot proliferation, growth, and rooting than that of the horizontal culture method regardless of the BA pre-treatment duration. The highest shoot multiplication was achieved through the vertical culture method with axillary bud explant, whereas the best shoot elongation and rooting was obtained using the vertical culture method with the apical bud explant. Apical bud explant was superior to axillary bud explant in ex vitro micro-cuttings and revealed a significant difference in shoot growth and root development. The above results suggest that explant position and culture method influence the efficiency of micropropagation for a rare and endangered plant Abeliophyllum distichum.

HOCl Oxidation-modified CT26 Cell Vaccine Inhibits Colon Tumor Growth in a Mouse Model

  • Zhou, Rui;Huang, Wen-Jun;Ma, Cong;Zhou, Yan;Yao, Yu-Qin;Wang, Yu-Xi;Gou, Lan-Tu;Yi, Chen;Yang, Jin-Liang
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.8
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    • pp.4037-4043
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    • 2012
  • Despite progress in elucidating mechanisms associated with colorectal cancer and improvement of treatment methods, it remains a frequent cause of death worldwide. New and more effective therapies are therefore urgently needed. Recent studies have shown that immunogenicity of whole ovarian tumor cells and subsequent T cell response were potentiated by oxidation modification with hypochlorous acid (HOCl) in vitro and ex vivo. These results prompted us to investigate the protective antitumor response with an HOCl treated CT26 colorectal cancer cell vaccine in an in vivo mouse model. Administration of HOCl modified vaccine triggered robust antitumor immunity to autologous tumor cells in mice and prolonged survival period significantly. In addition, increased necrosis and apoptosis were found in tumor tissue from the oxidation group. Interestingly, ELISPOT assays showed that specific T cell responses were not elicited in response to the immunizing cellular antigen, in contrast to raising sera antibody titer and antibody binding activity shown by ELISA assay and flow cytometry. Further evaluation of the mechanisms underlying HOCl modified vaccine mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results combined with previous studies suggest that HOCl oxidation modified whole cell vaccine has wide applicability as a cancer vaccine because it can target both T cell- and B cell-specific responses. It may thus represent a promising approach for the immunotherapy of colorectal cancer.

Morphogenetic Responses according to Media and Growth Regulators in vitro Culture of Eight Populus spp. (배지(培地) 및 생장(生長)호르몬에 따른 기내배양(器內培養)된 포푸라속(屬) 8개(個) 수종(樹種)의 기관형성반응(器官形成反應))

  • Kim, Chi Moon;Kwon, Ki Won;Moon, Heung Kyu;Lee, Jae Soon
    • Korean Journal of Agricultural Science
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    • v.14 no.2
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    • pp.205-212
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    • 1987
  • To examine the morphogenetic response, stem segments of 8 Populus spp. and 3 different explants of P. nigra var. italica were cultured on MS (Murashige and Skoog 1962) and WPM (Woody Plant Medium) medium containing various phytohormones. The results obtained were as follows: 1. Shoot regeneration and development from stem segment of 8 Populus spp. showed a quite difference according to the section and the species. All of the species of Leuce and Tacamahaca section did not form adventitious buds, while most of explants showed axillary or dormant bud elongation after 4 weeks. But P. nigra var. italica of Aigeiros section showed a successful adventitious bud formation (mean 5.4 buds per explant). 2. Leaf, petiole, and internode segment of P. nigra var. italica showed a quite differences according to media and ex plants upon the morphogenetic response. Adventitious bud formation from leaf was more abundant and readily initiated on the abaxial side than on the adaxial side. Mean number of 103 adventitious buds per explant was obtained from abaxial side of leaf segment cultured on WPM medium containing $0.2mg/{\ell}$ BAP for 5 weeks. 3. 2,4-D (2,4-dichlorophenoxy acetic acid) supplemented to media appeared to be negative upon the adventitious bud formation of P. nigra var. italica, while it promoted callus formation from all explants. Especially, NAA (${\alpha}$-naphtalene acetic acid) or NAA combination with BAP (6-benzylaminopurine) promoted root regeneration from the all explant of P. nigra var. italica in this study.

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Tooth whitening effect of toothpaste containing hydrogen peroxide (과산화수소가 배합된 제제의 치아미백효과에 관한 연구)

  • Ahn, Jae-Hyun;Kim, Ji-Hye;Kim, Jong-Hoon;Jang, Jong-Hwa;Oh, Yoon-Jong;Park, Yong-Duk
    • Journal of Korean society of Dental Hygiene
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    • v.14 no.1
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    • pp.101-108
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    • 2014
  • Objectives : The purpose of this study was to evaluate the tooth whitening effect of 0.74% and 2.80% hydrogen peroxide toothpastes and safety on tooth and gingival tissue. Methods : Toothpastes containing 0.74% and 2.80% hydrogen peroxide were evaluated. In in-vitro test, some additives (sodium metaphosphate, sodium pyrophosphate and titanium dioxide) were added to the toothpastes. Hydroxyapatite specimens (HAPs) were made and stained using modified Stookey's methods. HAPs were treated for 1 hour at shaking incubator and brushed for 1,000 times as 250 gF with each diluted toothpaste. Before and after color was measured by colorimeter. Using double blind method, 99 Korean with natural maxillary anterior teeth were selected and the initial brightness (baseline) was measured by SHADEEYE-EX. Based on this measurement they were crossly distributed into control group (0% hydrogen peroxide), test 1 (0.74% hydrogen peroxide) and test 2 (2.80% hydrogen peroxide). After 2 weeks, people of each group were provided toothpaste and told to use 3 times a day right after every meal for 3 minutes. The brightness of teeth was measured 3 times for every one month. Results : ${\Delta}L$ was statistically significant among three groups in shaking test. ${\Delta}L$ of two test groups was statistically significant compared with control group but not between each test group in brushing test. After using toothpaste for 3 months, test 1 group and test 2 group were 15.89% and 31.23% more whitened compared with control group respectively (p<0.05). Rate of more whitened person of each test group was 24.2% and 40.5% more than control group respectively (p<0.05). There was no difference in the hypersensitivity during 3 months using toothpastes and no side effect on teeth or gums. Conclusions : Toothpastes containing 0.74% and 2.80% hydrogen peroxide showed tooth whitening effect and both were safe enough to use for tooth whitening.

Plant Production from Desiccated Somatic Embryos of Acanthopanax chiisanensis (지리오가피 (Acanthopanax chiisanensis) 체세포배의 건조처리를 통한 식물체 증식)

  • Lee, Kang-Seop;Bang, Keuk-Soo;Choi, Yong-Eui;Ahn, Byung-Yong
    • Journal of Plant Biotechnology
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    • v.30 no.4
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    • pp.381-385
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    • 2003
  • An efficient method of plant regeneration from Acanthopanax chiisanensis somatic embryos was developed. Cotyledonary somatic embryos were obtained in liquid Murashige and Skoog (MS) medium from embryogenic cell suspension cultures. They were desiccated for 0 to 72 hr and then cultured on MS medium containing NAA, BA, GA$_3$, (0-0.5mg/L). The highest multiple shoots formation (100%) was obtained from 72 hr desiccated somatic embryos on ifs medium with 0.5mg/L NAA+0.5mg/L BA or 0.5 mg/L NAA+0.5mg/L BA+0.5mg/L GA$_3$ after 6 weeks culture. Plant conversion from multiple shoots was not high. The highest plant conversion from multiple shoots was obtained on 1/3MS medium with 1.0mg/L GA$_3$. Converted plantlets were transferred to ex vitro condition and the highest survival rate (70%) of the plantlets was obtained on plastic pots containing vermiculite and sand. These results indicate that micropropagation procedure can be applied for an efficient mass propagation of Acanthopanax chiisanensis.

Early caries detection using optical coherence tomography: a review of the literature (광간섭단층촬영술을 이용한 치아우식증의 발견)

  • Park, Young-Seok;Cho, Byeong-Hoon;Lee, Seung-Pyo;Shon, Won-Jun
    • Restorative Dentistry and Endodontics
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    • v.36 no.5
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    • pp.367-376
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    • 2011
  • Early detection of carious lesions increases the possibility of treatment without the need for surgical intervention. Optical coherence tomography (OCT) is an emerging three-dimensional imaging technique that has been successfully used in other medical fields, such as ophthalmology for optical biopsy, and is a prospective candidate for early caries detection. The technique is based on low coherence interferometry and is advantageous in that it is non-invasive, does not use ionizing radiation, and can render threedimensional images. A brief history of the development of this technique and its principles are discussed in this paper. There have been numerous studies on caries detection, which were mostly in vitro or ex vivo experiments. Through these studies, the feasibility of OCT for caries detection was confirmed. However, further research should be performed, including in vivo studies of OCT applications, in order to prove the clinical usefulness of this technique. In addition, some technological problems must be resolved in the near future to allow for the use of OCT in everyday practice.