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Molecular Cloning of Mutant cDNA of PU.1 Gene (PU.1 유전자(cDNA)의 인위적 변이체 클로닝)

  • 류종석;유시현
    • KSBB Journal
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    • v.10 no.5
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    • pp.499-509
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    • 1995
  • PU.1, a tissue-specific transcription activator, binds to a purine-rich sequence(5'-GAGGAA-3') called PU box. The PU.1 cDNA consists of an open reading frame of 816 nucleotides coding for 272 amino acids. The amino terminal end is highly acidic, while the carboxyl terminal end is highly basic. Transcriptional activation domain is located at the amino terminal end, while DNA binding domain is located at the carboxyl terminal end. Activation of PU.1 transcription factor is supposed to be accomplished by the phosphorylation of serine residue(s). There exist 22 serines in the PU.1. Five(the 41, 45, 132$.$133, and 148th) of the serines(plausible phosphorylation site by casein kinase II), are the primary targets of interest in elucidating the molecular mechanism(s) of the action of the PU.1 gene. In this study, PU.1 cDNA coding for the five serine residues(41th AGC, 45th AGC, 132$.$133th AGC$.$TCA, and 148th TCT), was mutated to alanine codon(41th GCC, 45th GCC, 132$.$133th GCC$.$GCA, and 1481h GCT), respectively, by Splicing-Overlapping-Extension(SOE) using Polymerase Chain Reaction(PCR). And each mutated cDNA fragments was ligated into pBluescript KS+ digested with HindIII and Xba I, to generate mutant clones named pKKS41A, pRKS45A, pMKS132$.$133A, and pMKS148A. The clones will be informative to study the "Structure and Function" of the immu-nologically important gene, PU.1.

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Structural Behavior of Composite Basement Wall According to Shear Span-to-Depth Ratio and FE Analysis Considering the Condition of Contact Surface (전단경간비에 따른 합성지하벽의 거동과 접촉면의 조건을 고려한 유한요소 해석)

  • Seo, Soo Yeon
    • Journal of the Korea institute for structural maintenance and inspection
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    • v.15 no.6
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    • pp.118-126
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    • 2011
  • The objective of this paper is to study the structural behavior of Composite Basement Wall (CBW) according to shear span-to-depth ratio through an experiment and predict the nonlinear behavior of CBW by using ADINA program widely has been being used for FE analysis. Especially, this study focuses on the part of CBW in which the Reinforced Concrete (RC) is under compression stress; At the region of CBW around each floor, RC part stresses by compressive force when lateral press by soil acts on the wall. The contact condition between RC wall and steel (H-Pile) including stud connector is main factor in the analysis since it governs overall structural behavior. In order to understand the structural behavior of CBW whose RC part is under compressive stress, an experimental work and finite element analysis were performed. Main parameter in the test is shear span-to-depth ratio. For simplicity in analysis, reinforcements were not modeled as a seperated element but idealized as smeared to concrete. All elements were modeled to have bi-linear relation of material properties. Three type of contact conditions such as All Generate Option (AGO), Same Element Group Option with Tie(SEGO-T) and Same Element Group Option with Not tie(SEGO-NT) were considered in the analysis. For each analysis, the stress flow and concentration were reviewed and analysis result was compared to test one. From the test result, CBW represented ductile behavior by contribution of steel member even if it had short shear span-to-depth ration which is close to "1". The global composite behavior of CBW whose concrete wall was under compressive stress could be predicted by using contact element in ADINA program. Especially, the modeling by using AGO and SEGO-T showed more close relation on comparing with test result.

Genome-Wide Association Analyses on Blood Pressure Using Three Different Phenotype Definitions

  • Park, Ji-Wan;Uhmm, Saan-Yong;Shin, Chol;Cho, Nam-H.;Cho, Yoon-Shin;Lee, Jong-Young
    • Genomics & Informatics
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    • v.8 no.3
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    • pp.108-115
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    • 2010
  • Hypertension is the most prevalent disease worldwide and is itself a risk factor for cerebral, cardiac, and renal diseases. The inconsistency of candidate genes suggested by previous genomewide association studies (GWASs) may be due to not only differences in study design and genetic or environmental background but also the difference in the power of analysis between continuous traits and discrete traits. We analyzed 352,228 single nucleotide polymorphisms (SNPs) in 8842 unrelated Koreans obtained from Ansan and Ansung cohorts. We performed a series of GWA analyses using three different phenotype models; young hypertensive cases (278 subjects) versus elderly normotensive controls (680 subjects); the upper 25% (2211 hypertensive cases) versus the lower 25% of the SBP distribution (2211 hypotensive controls); and finally SBP and DBP as continuous traits (8842 subjects). The numbers of young hypertensive cases and elderly normotensive controls were not large enough to achieve genomewide significance. The model comparing the upper 25% subjects to the lower 25% of subjects showed a power that was approximate to that of QTL analysis. Two neighboring SNPs of the ATP2B1 gene, rs17249754 (SBP, p=$2.53^{-10}$; DBP, p=$1.28{\times}10^{-8}$) and rs7136259 (SBP, p=$1.30{\times}10^{-9}$; DBP, p=$6.41{\times}10^{-8}$), were associated with both SBP and DBP. Interestingly, a SNP of the RPL6 gene, rs11066280, revealed a significant genomewide association with SBP in men only (p=$3.85{\times}10^{-8}$), and four SNPs located near the MAN2A1 gene showed a strong association with DBP only in elderly men aged 60-70 years (e.g., rs6421827, p=$4.86{\times}10^{-8}$). However, we did not observe any gene variant attaining genomewide significance consistently in the three phenotype models except for the ATP2B1 gene variants. In general, the association signal with blood pressure was stronger in women than in men. Genes identified in GWASs are expected to open the way for prevention, early diagnosis, and personalized treatment of hypertension.

Development of the computer program calculating the stress induced by various loads for buried natural gas pipeline (II) (매설 천연가스 배관의 제반하중에 의한 응력 계산용 프로그램 개발 (II))

  • Bang I.W.;Kim H.S.;Yang Y.C.;Kim W.S.;Oh K.W.
    • Journal of the Korean Institute of Gas
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    • v.2 no.2
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    • pp.26-33
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    • 1998
  • The thickness of buried gas pipeline is determined mainly with internal pressure and location factor according to the requirements of ANSI B3l.8. But the stress of buried gas pipeline is determined by not only internal stress but also external loads. The change of burying and environmental conditions, therefore, may result in increasing stress of pipeline. In order to avoid the decrease of safety degree resulting from change of environmental condition, the evaluation of stress level shall be necessary. The reliable equations have been developed for calculating stress of buried pipeline from internal pressure, earth load, vehicle load, ground subsidence. But they are very difficult to understand and use for non-specialist. For easy calculation of non-specialist, the new computer program to calculate stress of buried natural gas pipeline have been developed. The program can calculate maximum stress resulted from earth load, vehicle load, thermal load, four type ground subsidence. The stress is calculated by the equations and extrapolation of the graph resulted from FEM. In this paper, as the series of paper I, the operating method and the functions of the program is explained.

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Studies on In Vitro Culture, Freezing and Transfer of Korean Native Cattle Embryos Fertilized In Vitro H. Factors Affecting on Survival Rate of Frozen-Thawed Korean Native Cattle Embryos Fertilized In Vitro (한우 체외수정란의 체외배양, 동결보존 및 이식에 관한 연구 II. 한우 체외수정란의 동결 및 융해 후 생존율에 영향을 미치는 요인)

  • 김일화;손동수;이호준;최선호;양병철;이광원;장인호
    • Journal of Embryo Transfer
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    • v.11 no.2
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    • pp.125-135
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    • 1996
  • The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6$^{\circ}C$ to -35$^{\circ}C$ at -0.3$^{\circ}C$ or -O.6$^{\circ}C$ /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3$^{\circ}C$ /min and -0.6$^{\circ}C$ /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3$^{\circ}C$ /min(63.6%) than in -0.6$^{\circ}C$ /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3$^{\circ}C$ /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

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Role of $NF-_{{\kappa}B}$ Binding Sites in the Regulation of Inducible Nitric Oxide Synthase by Tyrosine Kinase

  • Ryu, Young-Sue;Hong, Jang-Hee;Lim, Jong-Ho;Bae, So-Hyun;Ahn, Ihn-Sub;Seok, Jeong-Ho;Lee, Jae-Heun;Hur, Gang-Min
    • The Korean Journal of Physiology and Pharmacology
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    • v.5 no.1
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    • pp.55-63
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    • 2001
  • In macrophages, lipopolysaccharide (LPS) alone or in combination with $interferon-{\gamma}\;(IFN-{\gamma})$ has been shown to release a nitric oxide (NO) through the increase of the transcription of the inducible nitric oxide synthase (iNOS) gene. To investigate the exact intracellular signaling pathway of the regulation of iNOS gene transcription by LPS plus $IFN-{\gamma},$ the effects of protein tyrosine kinase (PTK) inhibitor and protein kinase C (PKC) inhibitors on NO production, iNOS mRNA expression, nuclear $factor-_{\kappa}B\;(NF-_{\kappa}B)$ binding activity and the promoter activity of iNOS gene containing two $NF-_{\kappa}B$ sites have been examined in a mouse macrophage RAW 264.7 cells. LPS or $IFN-{\gamma}$ stimulated NO production, and their effect was enhanced synergistically by mixture of LPS and $IFN-{\gamma}.$ The PTK inhibitor such as tyrphostin reduced LPS plus $IFN-{\gamma}-induced$ NO production, iNOS mRNA expression and $NF-_{\kappa}B$ binding activity. In contrast, PKC inhibitors such as H-7, Ro-318220 and staurosporine did not show any effect on them. In addition, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that tyrphostin inhibited the iNOS promoter activity through the $NF-_{\kappa}B$ binding site, whereas PKC inhibitors did not. Taken together, these suggest that PTK, but not PKC pathway, is involved in the regulation of the iNOS gene transcription through the $NF-_{\kappa}B$ sites of iNOS promoter in RAW 264.7 macrophages by LPS plus $IFN-{\gamma}$.

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Generation of $CD2^+CD8^+$ NK Cells from c-$Kit^+$ Bone Marrow Cells in Porcine

  • Lim, Kyu-Hee;Han, Ji-Hui;Roh, Yoon-Seok;Kim, Bum-Seok;Kwon, Jung-Kee;You, Myoung-Jo;Han, Ho-Jae;Ejaz, Sohail;Kang, Chang-Won;Kim, Jong-Hoon
    • The Korean Journal of Physiology and Pharmacology
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    • v.16 no.3
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    • pp.167-174
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    • 2012
  • Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-$kit^+$ bone marrow cells (c-$kit^+$ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-$kit^+$ BM cells that give rise to $CD2^+CD8^+$ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-$kit^+$ BM cells than those of controls. And, we compared the ability of the cytotoxicity of $CD2^+CD8^+$ NK cells differentiated by cytokines from c-$kit^+$ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100 : 1 for 4 h once a week. In results, $CD2^+CD8^+$ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-$kit^+$ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-$kit^+$ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

The Verification of Photoplethysmography Using Green Light that Influenced by Ambient Light (녹색광을 이용한 반사형 광용적맥파측정기의 주변광 간섭시 신호측정)

  • Chang, K.Y.;Ko, H.C.;Lee, J.J.;Yoon, Young Ro
    • Journal of Biomedical Engineering Research
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    • v.35 no.5
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    • pp.125-131
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    • 2014
  • The purpose of this study is to verify the utility of reflected photoplethysmography sensor using two green light emitting diodes that influenced by ambient light. Recently it has been studied that green light emitting diode is suitable for light source of reflected photoplethysmography sensor at low temperature and high temperature. Another study showed that, green light is better for monitoring heart rate during motion than led light. However, it has a bad characteristic about ambient light noise. To verify the utility of reflected photoplethysmography sensor using green light emitting diode, this study measures the photoplethysmography signal that is distorted by ambient light and will propose a solution. This study has two parts of research method. One is measurement system that composed sensor and board. The sensor is made up PE-foam and Non-woven fabric for flexible sensor. The photoplethysmography signal is measured by measurement board that composed high-pass filter, low-pass filter and amplifier. Ambient light source is light bulb and white light emitting diode that has three steps brightness. Photoplethysmography signal is measured with lead II electrocardiography signal at the same time and it is measured at the finger and radial artery for 1 minute, 1000 Hz sampling rate. The lead II electrocardiography signal is a standard signal for heart rate and photoplethysmography signal that measured at the finger is a standard signal for waveform. The test is repeated 3 times using three sensor. The data is processed by MATLAB to verify the utility by comparing the correlation coefficient score and heart rate. The photoplethysmography sensor using two green light emitting diodes is shown better utility than using one green light emitting diode and red light emitting diode at the ambient light. The waveform and heart rate that measured by two green light emitting diodes are more identical than others. The amount of electricity used is less than red light emitting diode and error peak detectability factor is the lowest.

Structural Capacity Evaluation of System Scaffolding using X-Type Advanced Guardrail (교차가새형 선행 안전난간을 적용한 시스템비계의 구조 성능 평가)

  • Park, J.D.;Lee, H.S.;Shin, W.S.;Kwon, Y.J.;Park, S.E.;Yang, S.S.;Jung, K.
    • Journal of the Korean Society of Safety
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    • v.35 no.5
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    • pp.49-58
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    • 2020
  • In domestic construction sites, when installing steel pipe scaffolding and system scaffolding, the guardrails are installed after the installation of the work platforms. This conventional guardrail system (CGS) is always exposed to the risk of falls because the safety railing is installed later. In order to prevent fall disasters during erecting and dismantling scaffolds, it is necessary to introduce the advanced guardrail system (AGS) which installs railings in advance of climbing onto a work platform. For the introduction of the AGS, the structural performance of the system scaffolding applying the CGS and the AGS was compared and evaluated. The structural analysis of the system scaffold (height: 31 m and width: 27.4 m) with AGS confirmed that structural safety was ensured because the maximum stress of each element of the system scaffolding satisfies the allowable stress of each element. As a result of performance comparison of CGS and AGS for each element, the combined stress ratio of vertical posts in AGS was 6.4% lower than that of CGS. In addition, in the case of ledger and transom, the combined stress ratios of AGS and CGS were almost the same. The compression test of the assembled system scaffolding (three-storied, 1 bay) showed that the AGS had better performance than the CGS by 9.7% (8.91 kN). The cross bracing exceeds the limit on slenderness ratio of codes for structural steel design. But the safety factor for the compressive load of the cross bracing was evaluated as meeting the design criteria by securing 3 or more. In actual experiments, it was confirmed that brace buckling did not occur even though the overall scaffold was buckled. Therefore, in the case of temporary structures, it was proposed to revise the standards for limiting on slenderness ratio of secondary or auxiliary elements to recommendations. This study can be used as basic data for the introduction of AGS for installing guardrails in advance at domestic construction sites.

Use Impacts of Trail and Campsite on Natural Environments in Mt. Palgong Natural Park (팔공산자연공원에서의 등산로 및 야영장 이용이 자연환경에 미치는 영향)

  • 권태호
    • Journal of the Korean Institute of Landscape Architecture
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    • v.17 no.3
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    • pp.21-34
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    • 1990
  • Trail and campground deteriorations and their environmental changes of soil and vegetation were studied in Tonghwa -Sa district of Mt. Palgong Natural Park through 1988 into 1989. The results are summarized as follows : 1. The bare width of trail as one of the trail condition factor was significantly greater on the more heavily used trail. Deterioration types of trail which had higher frequency for a total of 45 observations were rock exposure(42%), trail deepening(40%) and root exposure(18%) in the high order. And also sound type on which the trail was not deteriorated at all amounted to 33% of all observations. 2. Ecological changes of soil and vegatation of trailsides were not found at a uniform tendency except that soil hardness was slightly decreased from trail edge to the forest, but they could be grouped by the type of user\`s behavior and site conditions. Use impacts on the natural environment of trailside in Mt. Palgong are still far from the severe harmulness. 3. Worse damages to compground condition were appeared in Bawi-gol than Susu-gol campsite. Types of mechanically damaged trees were tree with exposed root(63% ), scared tree(43% ), mutilated tree(30 % ) and felled tree in highly frequent order. And diameter increments of trees in campsites were oppressed by the campers. 4. Tree damage types and their frequencies could be basic as a means of which grasp the bounds of user's impacts. User's disturbance on campsite extended to the distance of 70∼90m from the core of campsite at Pawi-gol and 20∼30m at Susu-gol respectively. As the tolerant trees to use impact, Lespedeza maximowiczii, Rhus trichocarpa, Acer palmatum, Rubus crataegifolius and Celastrus orbiculatus were considered.

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