• Title/Summary/Keyword: cell yield

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The Synthesis of the Stable IVDU Derivative for Imaging HSV-1 TK Expression (체내 안정형 HSV1-tk (Herpes Simplex Virus Type-1 Thymidine Kinase) 영상용 IVDU 유도체의 합성)

  • Kim, Eun-Jung;Choi, Tae-Hyun;Ahn, Soon-Hyuk;Kim, Byoung-Soo;Park, Hyun;Cheon, Gi-Jeong;Rhee, Hak-June;An, Gwang-Il
    • Nuclear Medicine and Molecular Imaging
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    • v.43 no.5
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    • pp.478-486
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    • 2009
  • Purpose: 5-iododeoxyuridine analogues have been exclusively developed for the potential antiviral and antitumor therapeutic agents. In this study, we synthesized carbocyclic radioiododeoxyuridineanalogue (ddIVDU) and carbocyclic intermediate as efficient carbocyclic radiopharmaceuticals. Materials and Methods: The synthesis is LAH reduction, hetero Diels-Alder reaction as key reactions including Pd(0)-catalyzed coupling reaction together with organotin. MCA-RH7777 (MCA) and MCA-tk (HSV1-tk positive) cells were treated with various concentration of carbocyclic ddIVDU, and GCV. Cytotoxicity was measured by the MTS methods. For in vitro uptake study, MCA and MCA-tk cells were incubated with 1uCi of [$^{125}I$]carbocyclic ddIVDU. Accumulated radioactivity was measured after various incubation times. Results: The synthesis of ddIVDU and precursor for radioiodination were achieved from cyclopentadiene in good overall yield, respectively. The radioiododemetallation for radiolabeling gave more than 80% yield with > 95% radiochemical purity. GCV was more toxic than carbocyclic ddIVDU in MCA-tk cells. Accumulation of [$^{125}I$]carbocyclic ddIVDU was higher in MCA-tk cells than MCA cells. Conclusion: Biological data reveal that ddIVDU is stable in vitro, less toxic than ganciclovir (GCV), and selective in HSV1-tk expressed cells. Thus, this new carbocyclic nucleoside, referred to in this paper as carbocyclic 2',3'-didehydro-2',3'-dideoxy-5- iodovinyluridine (carbocyclic ddIVDU), is a potential imaging probe for HSV1-tk.

Improving Corsican pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology and germination

  • Wtpsk, Senarath;Shaw, D.S.;Lee, Kui-Jae;Lee, Wang-Hyu
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2003.04a
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    • pp.61-62
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    • 2003
  • Clonal propagation of high-value forest trees through somatic embryogenesis (SE) has the potential to rapidly capture the benefits of breeding or genetic engineering programs and to improve raw material uniformity and quality. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Several factors limit commercialization of SE for Corsican pine, including low initiation rates, low culture survival, culture decline causing low or no embryo production, and inability of somatic embryos to fully mature, resulting in low germination and reduced vigour of somatic seedlings. The objective was to develop a Corsican pine maturation medium that would produce cotyledonary embryos capable of germination. Treatments were arranged in a completely randomized design. Data were analyzed by analysis of variance, and significant differences between treatments determined by multiple range test at P=0.05. Corsican pine (Pinus nigra var. maritima) cultures were initiated on modified !P6 medium. Modifications of the same media were used for culture multiplication and maintenance. Embryogenic cultures were maintained on the same medium semi solidified with 2.5 g/l Gelrite. A maturation medium, capable of promoting the development of Corsican pine somatic embryos that can germinate, is a combination of iP6 modified salts, 2% maltose, 13% polyethylene glycol (PEG), 5 mg!l abscisic acid (ABA), and 2.5 g/l Gelrite. After initiation and once enough tissue developed they were grown in liquid medium. Embryogenic cell suspensions were established by adding 0.951.05 g of 10- to 14-day-old semisolid-grown embryogenic tissue to 9 ml of liquid maintenance media in a 250ml Erlenmeyer flask. Cultures were then incubated in the dark at 2022$^{\circ}$C and rotated at 120 rpm. After 2.53 months on maturation medium, somatic embryos were selected that exhibited normal embryo shape. Ten embryos were placed horizontally on 20 ml of either germination medium ($\frac{2}{1}$strength Murashige and Skoog (1962) salts with 2.5 g/l activated charcoal) or same medium with copper sulphate adjusted to 0.25 mg/1 to compensate for copper adsorption by activated carbon. 2% and 4% maltose was substituted by 7.5% and 13% PEG respectively to improve the yield of the embryos. Substitution of' maltose with PEG was clearly beneficial to embryo development. When 2% of the maltose was replaced with 7.5% PEG, many embryos developed to large bullet-shaped embryos. At latter stages of development most embryos callused and stopped development. A few short, barrel-shaped cotyledonary embryos formed that were covered by callus on the sides and base. When 4% of the maltose was removed and substituted with 13% PEG, the embryos developed further, emerging from the callus and increasing yield slightly. Microscopic examination of the cultures showed differing morphologies, varying from mostly single cells or clumps to well-formed somatic embryos that resembled early zygotic embryos only liquid cultures with organized early-stag. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also tested. Seedling conversion and growth were highly related to the quality of the germinant at the time of planting. Germinants with larger shoots, longer, straighter hypocotyls and longer roots performed best. When mature zygotic embryos germinate the root emerges, before or coincident with the shoot. In contrast, somatic embryos germinate in reverse sequence, with the cotyledons greening first, then shoot emergence and then, much later, if at all, the appearance of the root. Somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed.

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Development of Radiation Image Sensor using Heterojunction (이종접합을 이용한 방사선 영상 센서 개발)

  • Kim, Young-Bin;Yun, Min-Seok;Kim, Min-Woo;Jung, Suk-Hee;Kim, Yoon-Suk;Oh, Kyung-Min;Nam, Sang-Hee;Park, Ji-Koon
    • Journal of the Korean Society of Radiology
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    • v.3 no.3
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    • pp.27-35
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    • 2009
  • In this study, the basic research verifying possibility of applications as radiology image sensor in Digital Radiography was performed, the radiology image sensor was fabricated using double layer technique tio decrease dark current. High efficiency material in substitution for a-Se have been studied as a direct method of imaging detector in Digital Radiography to decrease dark current by using Hetero junction already used as solar cell, semiconductor. Particle-In-Binder method is used to fabricate radiology image sensor because it has a lot of advantages such as fabrication convenient, high yield, suitability for large area sensor. But high leakage current is one of main problem in PIB method. To make up for the weak points, double layer technique is used, and it is considered that high efficient digital radiation sensor can be fabricated with easy and convenient process. In this study, electrical properties such as leakage current, sensitivity is measured to evaluate double layer radiation sensor material.

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Linkage Positions of Oligosaccharides by Low Energy Collision Tandem Mass Spectrometry: Effect of the Addition of Metal Cations (저에너지 충돌 탄뎀 질량분석법을 이용한 올리고당의 연결부위 연구: 금속양이온의 첨가가 미치는 영향)

  • Yoo Yoon, Eunsun
    • Journal of the Korean Chemical Society
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    • v.40 no.8
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    • pp.557-564
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    • 1996
  • FAB CAD MS/MS(Fast Atom Bombardment Collision Activated Dissociation Mass Spectrometry/Mass Spectrometry) was used to study different degree of bond stability according to the linkage positions of alkali cationized $(Na^+, Li^+, K^+, NH_4^+)$ stereoisomeric and synthetic oligosaccharides. The alkali metal cations were much more stable, requiring over -40 eV of collision energy vs. only -10 eV for the protonated forms. Of the cations, the potassium cationized trisaccharides were more stable than the others. They would not yield fragment ions under the conditions of collision available in triple quadrupole. Other cationized species exhibited decreasing stability in the series $Nap^+>Li^+>NH_4^+$ using 0.8 mTorr argon pressure in the collision cell. Metal cations of the oligosaccharides maintained charge principally on the amino sugar as shown by shift of all the fragment ions containing the amino sugar. The reason for the higher stability of the metal cationized form is the formation of crown ether-like bond around metal cations, N-acetyl group on GlcNAc and oxygens on fucose moiety. Depending on the metal sizes and the conformation of linkage-isomeric region, cationized species gave linkage dependent fragment patterns and exhibited stability in the series 1-6 > 1-4 > 1-3 linkage.

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Determination of the Temperature Increasing Value of Seedling Nursery Period for Oryza2000 Model to Applicate Grid Weather Data (Oryza2000 모형 활용을 위한 육묘기 보온 상승온도 결정)

  • Kim, Junhwan;Sang, Wangyu;Shin, Pyeong;Baek, Jaekyeong;Kwon, Dongwon;Lee, Yunho;Cho, Jung-Il;Seo, Myungchul
    • Korean Journal of Agricultural and Forest Meteorology
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    • v.22 no.1
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    • pp.20-25
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    • 2020
  • Spatial simulation of crop growth often requires application of management conditions to each cell. In particular, it is of great importance to determine the temperature conditions during the nursery period for rice seedlings, which would affect heading date projections. The objective of this study was to determine the value of TMPSB, which is the parameter of ORYZA2000 model to represent temperature increase under a plastic tunnel during the rice seedling periods. Candidate values of TMPSB including 0℃, 2℃, 5℃, 7℃ and 9℃ were used to simulate rice growth and yield. Planting dates were set from mid-April to mid-June. The simulations were performed at four sites including Cheorwon, Suwon, Seosan, and Gwangju where climate conditions at rice fields common in Korea can be represented. It was found that the TMPSB values of 0℃ and 2℃ resulted in a large variation of heading date due to low temperature occurred in mid-April. When the TMPSB value was >7℃, the variation of heading date was relatively small. Still, the TMPSB value of 5℃ resulted in the least variation of heading date for all the planting dates. Our results suggested that the TMPSB value of 5℃ would help reasonable assessment of climate change impact on rice production when high resolution gridded weather data are used as inputs to ORYZA2000 model over South Korea.

Culture Conditions of E. coli CK1092 for the Production of 2,3-Dihydroxybiphenyl Dioxygenase (2,3-Dihydroxybiphenyl Dioxygenase 생산을 위한 E. coli CK1092의 배양조건)

  • Lee, Jung-Young;Kim, Youngsoo;Lee, Ki-Sung;Min, Kyung-Hee;Kim, Young-Chang;Kim, Chi-Kyung;Lim, Jai-Yun
    • Korean Journal of Microbiology
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    • v.34 no.1_2
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    • pp.20-25
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    • 1998
  • To obtain higher yield of 2,3-dihydroxybiphenyl(2,3-DHBP) dioxygenase by recombinant E. coli CK1092 carrying pcbC gene of Pseudomonas sp. P20, the environmental and physiological factors were investigated and the cultural conditions using jar fermentor were studied. E. coli CKl092 was grown in LB medium supplemented with 2% sucrose, as a basal medium. The effect of various metal ions on the enzyme production was investigated. In particular, the enzyme production increased in the presence of $Fe^{3+}$ and $Fe^{2+}$, and showed the maxium at the concentration of $10^{-5}M$. The enzyme production was increased by 55% in the medium containing $Fe^{3+}$ ($10^{-5}M$) ion. The optimal temperature and initial pH for cell growth were $37^{\circ}C$ and 7.0, respectively. In the culture using a fermentor at $37^{\circ}C$, the optimal conditions for the enzyme production were obtained at the initial pH 7.0, 1 v/v/m of aeration rate, 200 rpm of agitation speed. It was found that enzyme activity was higher when cultivated without pH control than with pH control.

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Development of bombesin peptide kit for prostate cancer diagnosis and treatment using 64Cu/177Lu.

  • Kim, Mi Hyun;Kim, Min Hwan;Kim, Kwang Il;Kim, Jung Young;Lee, Tae Sup;Kang, Joo Hyun;Lee, Kyo Chul;Lee, Yong Jin
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.3 no.2
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    • pp.59-64
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    • 2017
  • It has been reported that $^{64}Cu$ was radiolabeled with bombesin (BBN) peptide binding to the gastrin releasing peptide receptor expressed in human prostate cancer cells (PC3), confirming tumor target efficacy in mouse model. In this study, we developed the kit for the diagnosis and treatment of prostate cancer that can be used clinically using bombesin peptide available of $^{64}Cu$ and $^{177}Lu$ radioisotope labeling. The NODAGA-galacto-BBN peptide containing the NODAGA chelator and galactose was dispensed into a sterilized glass vial and lyophilized to prepare a kit. The stability of the kit after long-term storage in the $4^{\circ}C$ cold chamber and the radiolabeling efficiency after $^{64}Cu$ or $^{177}Lu$ labeling were confirmed by thin layer chromatography. When labeling with $^{64}Cu$ at the initial stage of storage, labeling efficiency of NODAGA-galacto-BBN peptide kit was over 96%, labeling efficiency was over 90% when $^{177}Lu$ was labeled. At 11 months after storage, the radiolabeling efficiency of kit against $^{64}Cu$ and $^{177}Lu$ was each over 95% and 90%. The cell viability was significantly reduced in the $^{177}Lu$-NODAGA-galacto-BBN treated group compared with the control and $^{177}Lu$ alone treated group in clonogenic assay. In conclusion, the NODAGA-galacto-BBN kit prepared by the lyophilization showed high stability over time and high yield of radioisotope labeling. Also $^{177}Lu$-NODAGA-galacto-BBN confirmed high cytotoxicity to prostate cancer cells. Therefore, the NODAGA-galacto-bombesin kit is expected to be useful for the diagnosis and treatment of prostate cancer patients.

Physiological activities of Lespedeza cuneata extracts (야관문 추출물의 생리 활성)

  • Park, Hye-Mi;Hong, Joo-Heon
    • Food Science and Preservation
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    • v.21 no.6
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    • pp.844-850
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    • 2014
  • This study was conducted to examine the physiological activities of Lespedeza cundata extracts. The extraction yield of 50% ethanol extract (17.60%) was higher than that of hot water extract (12.60%). The total phenolic and total flavonoid contents of the 50% ethanol extract were 242.26 mg/g and 160.73 mg/g, respectively. The DPPH radical scavenging activities of the hot water and 50% ethanol extracts were 92.07% and 96.38%, respectively. The superoxide radical scavenging activities of hot water and 50% ethanol extracts on $250{\sim}1,000{\mu}g/mL$ were 54.89~85.68% and 44.50~94.46%, respectively. The tyrosinase inhibition activity of the 50% ethanol extract at $1,000{\mu}g/mL$ (63.31%) was the highest. The nitrite scavenging activity of the 50% ethanol extract was higher than that of the hot water extract. The nitric oxide production of 50% ethanol extract ($7.15{\sim}20.61{\mu}M$) improved with an increase in the treatment concentration. The hot water and 50% ethanol extracts at $1,000{\mu}g/mL$ inhibited the proliferation of the cancer cell lines A549, HeLa, Hep3B, and Sarcoma180. There results suggest that the 50% ethanol Lespedeza cuneata extracts may be useful as a functional food material in the food industry.

Exploiting cDNA Microarray-Based Approach Combined with RT-PCR Analysis to Monitor the Radiation Effect: Antioxidant Gene Response of ex vivo Irradiated Human Peripheral Blood Lymphocyte

  • Sung, Myung-Hui;Jun, Hyun-Jung;Hwang, Seung-Yong;Hwang, Jae-Hoon;Park, Jong-Hoon;Han, Mi-Young;Lee, U-Youn;Park, Eun-Mi;Park, Young-Mee
    • Environmental Mutagens and Carcinogens
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    • v.22 no.3
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    • pp.142-148
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    • 2002
  • Although ionizing radiation (IR) has been used to treat the various human cancers, IR is cytotoxic not only to cancer cells but to the adjacent normal tissue. Since normal tissue complications are the limiting factor of cancer radiotherapy, one of the major concerns of IR therapy is to maximize the cancer cell killing and to minimize the toxic side effects on the adjacent normal tissue. As an attempt to develop a method to monitor the degree of radiation exposure to normal tissues during radiotherapy, we investigated the transcriptional responses of human peripheral blood lymphocytes (PBL) following IR using cDNA microarray chip containing 1,221 (1.2 K) known genes. Since conventional radiotherapy is delivered at about 24 h intervals at 180 to 300 cGy/day, we analyzed the transcriptional responses ex-vivo irradiated human PBL at 200 cGy for 24 h-period. We observed and report on 1) a group of genes transiently induced early after IR at 2 h, 2) of genes induced after IR at 6 h, 3) of genes induced after IR at 24 h and on 4) a group of genes whose expression patters were not changed after IR. Since Biological consequences of IR involve generation of various reactive oxygen species (ROS) and thus oxidative stress induced by the ROS is known to damage normal tissues during radiotherapy, we further tested the temporal expression profiles of genes involved in ROS modulation by RT-PCR. Specific changes of 6 antioxidant genes were identified in irradiated PBL among 9 genes tested. Our results suggest the potential of monitoring post-radiotherapy changes in temporal expression profiles of a specific set of genes as a measure of radiation effects on normal tissues. This type of approach should yield more useful information when validated in in vivo irradiated PBL from the cancer patients.

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Validation of Methods for Isolation and Culture of Alpaca Melanocytes: A Novel Tool for In vitro Studies of Mechanisms Controlling Coat Color

  • Bai, Rui;Sen, Aritro;Yu, Zhihui;Yang, Gang;Wang, Haidong;Fan, Ruiwen;Lv, Lihua;Lee, Kyung-Bon;Smith, George W;Dong, Changsheng
    • Asian-Australasian Journal of Animal Sciences
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    • v.23 no.4
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    • pp.430-436
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    • 2010
  • The objective of the present studies was to develop and validate a system for isolation, purification and extended culture of pigment-producing cells in alpaca skin (melanocytes) responsible for coat color and to determine the effect of alpha melanocyte stimulating hormone treatment on mRNA expression for the melanocortin 1 receptor, a key gene involved in coat color regulation in other species. Skin punch biopsies were harvested from the dorsal region of 1-3 yr old alpacas and three different enzyme digestion methods were evaluated for effects on yield of viable cells and attachment in vitro. Greatest cell yields and attachment were obtained following dispersion with dispase II relative to trypsin and trypsin-EDTA treatment. Culture of cells in medium supplemented with basic fibroblast growth factor, bovine pituitary extract, hydrocortisone, insulin, 12-O-tetradecanolphorbol-13-acetate and cholera toxin yielded highly pure populations of melanocytes by passage 3 as confirmed by detection of tyrosinase activity and immunocytochemical localization of melanocyte markers including tyrosinase, S-100 and micropthalmia-associated transcription factor. Abundance of mRNA for tyrosinase, a key enzyme in melanocyte pigment production, was maintained through 10 passages showing preservation of melanocyte phenotypic characteristics with extended culture. To determine hormonal responsiveness of cultured melanocytes and investigate regulation of melanocortin 1 receptor expression, cultured melanocytes were treated with increasing concentrations of ${\alpha}$-melanocyte stimulating hormone. Treatment with ${\alpha}$-melanocyte stimulating hormone increased melanocortin receptor 1 mRNA in a dose dependent fashion. The results demonstrated culture of pure populations of alpaca melanocytes to 10 passages and illustrate the potential utility of such cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in fiber-producing species.