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Production and Purification of Single Chain Human Insulin Precursors with Various Fusion Peptides

  • Cho, Chung-Woo;Park, Sun-Ho;Nam, Doo-Hyun
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.6 no.2
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    • pp.144-149
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    • 2001
  • For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

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Performance of steel beams at elevated temperatures under the effect of axial restraints

  • Liu, T.C.H.;Davies, J.M.
    • Steel and Composite Structures
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    • v.1 no.4
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    • pp.427-440
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    • 2001
  • The growing use of unprotected or partially protected steelwork in buildings has caused a lively debate regarding the safety of this form of construction. A good deal of recent research has indicated that steel members have a substantial inherent ability to resist fire so that additional fire protection can be either reduced or eliminated completely. A performance based philosophy also extends the study into the effect of structural continuity and the performance of the whole structural totality. As part of the structural system, thermal expansion during the heating phase or contraction during the cooling phase in most beams is likely to be restrained by adjacent parts of the whole system or sub-frame assembly due to compartmentation. This has not been properly addressed before. This paper describes an experimental programme in which unprotected steel beams were tested under load while it is restrained between two columns and additional horizontal restraints with particular concern on the effect of catenary action in the beams when subjected to large deflection at very high temperature. This paper also presents a three-dimensional mathematical modelling, based on the finite element method, of the series of fire tests on the part-frame. The complete analysis starts with an evaluation of temperature distribution in the structure at various time levels. It is followed by a detail 3-D finite element analysis on its structural response as a result of the changing temperature distribution. The principal part of the analysis makes use of an existing finite element package FEAST. The effect of columns being fire-protected and the beam being axially restrained has been modelled adequately in terms of their thermal and structural responses. The consequence of the beam being restrained is that the axial force in the restrained beam starts as a compression, which increases gradually up to a point when the material has deteriorated to such a level that the beam deflects excessively. The axial compression force drops rapidly and changes into a tension force leading to a catenary action, which slows down the beam deflection from running away. Design engineers will be benefited with the consideration of the catenary action.

Antiarrhythmic effects of ginsenoside Rg2 on calcium chloride-induced arrhythmias without oral toxicity

  • Gou, Dongxia;Pei, Xuejing;Wang, Jiao;Wang, Yue;Hu, Chenxing;Song, Chengcheng;Cui, Sisi;Zhou, Yifa
    • Journal of Ginseng Research
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    • v.44 no.5
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    • pp.717-724
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    • 2020
  • Background: Malignant arrhythmias require drug therapy. However, most of the currently available antiarrhythmic drugs have significant side effects. Ginsenoside Rg2 exhibits excellent cardioprotective effects and appears to be a promising candidate for cardiovascular drug development. So far, the oral toxicity and antiarrhythmic effects of Rg2 have not been evaluated. Methods: Acute oral toxicity of Rg2 was assessed by the Limit Test method in mice. Subchronic oral toxicity was determined by repeated dose 28-day toxicity study in rats. Antiarrhythmic activities of Rg2 were evaluated in calcium chloride-induced arrhythmic rats. Antiarrhythmic mechanism of Rg2 was investigated in arrhythmic rats and H9c2 cardiomyocytes. Results: The results of toxicity studies indicated that Rg2 exhibited no single-dose (10 g/kg) acute oral toxicity. And 28-day repeated dose treatment with Rg2 (1.75, 3.5 and 5 g/kg/d) demonstrated minimal, if any, subchronic toxicity. Serum biochemical examination showed that total cholesterol in the high-dose cohort was dramatically decreased, whereas prothrombin time was increased at Day 28, suggesting that Rg2 might regulate lipid metabolism and have a potential anticoagulant effect. Moreover, pretreatment with Rg2 showed antiarrhythmic effects on the rat model of calcium chloride induced arrhythmia, in terms of the reduced duration time, mortality, and incidence of malignant arrhythmias. The antiarrhythmic mechanism of Rg2 might be the inhibition of calcium influx through L-type calcium channels by suppressing the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Conclusion: Our findings support the development of Rg2 as a promising antiarrhythmic drug with fewer side effects for clinical use.

The Effect of Serum Obtained before and after Treatment for Endometriosis on in vitro Fertilization Rate of Mouse Oocyte (자궁내막증 치료 전후 환자의 혈청이 생쥐 난자의 수정률에 미치는 영향)

  • Kim, Kie-Suck;Min, Bu-Kie;Rhee, Hee-Sub;Hong, Kie-Youn;Lee, Sun-Young;Park, Heon-Jin;Kim, Heung-Gon
    • Clinical and Experimental Reproductive Medicine
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    • v.26 no.3
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    • pp.369-375
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    • 1999
  • Objective: To evaluate the effect of serum obtained before and after treatment for endometriosis on in vitro fertilization and development of two cell mouse embryo. Design: Pretreatment and posttreatment comparoson of fertilization of mouse oocyte and embryo development in serum supplement from patients with endometriosis; result were compared using Stuent T-test analysis. Method: Infertility Clinic, Department of Obstetrics and Gynecology, Collage of Medicine, Won kwang university, Korea. Patients was chosed eleven consecutive women with endometriosis. Interventions was all patient underwent laparoscopic or conservative surgery. This was followed by a 6-month course of burserelin acetate $900{\mu}g/d$. Main outcome was measured total number of fertilization and embryo that was fertilization after 24 hours and reached blastocyst stage after 72 hours of incubation were compared before and after treatment. Result: Before treatment, 47% of the oocyte were fertilized and 31% of the embryo reached blastocyst stage. After treatment, Significantly more fertilized and Significantly more embryo developed to blastocyst on the stage I and II of endometriosis. Conclusion: The fertilization and embryo toxicity of serum samples from patients with endometriosis is lost after treatment.

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Fabrication of a Pressure Difference Type Gas Flow Sensor using ICP-RIE Technology (ICP-RIE 기술을 이용한 차압형 가스유량센서 제작)

  • Lee, Young-Tae;Ahn, Kang-Ho;Kwon, Yong-Taek;Takao, Hidekuni;Ishida, Makoto
    • Journal of the Semiconductor & Display Technology
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    • v.7 no.1
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    • pp.1-5
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    • 2008
  • In this paper, we fabricated pressure difference type gas flow sensor using only dry etching technology by ICP-RIE(inductive coupled plasma reactive ion etching). The sensor's structure consists of a common shear stress type piezoresistive pressure sensor with an orifice fabricated in the middle of the sensor diaphragm. Generally, structure like diaphragm is fabricated by wet etching technology using TMAH, but we fabricated diaphragm by only dry etching using ICP-RIE. To equalize the thickness of diaphragm we applied insulator($SiO_2$) layer of SOI(Si/$SiO_2$/Si-sub) wafer as delay layer of dry etching. Size of fabricated diaphragm is $1000{\times}1000{\times}7\;{\mu}m^3$ and overall chip $3000{\times}3000{\times}7\;{\mu}m^3$. We measured the variation of output voltage toward the change of gas pressure to analyze characteristics of the fabricated sensor. Sensitivity of fabricated sensor was relatively high as about 1.5mV/V kPa at 1kPa full-scale. Nonlinearity was below 0.5%F.S. Over-pressure range of the fabricated sensor is 100kPa or more.

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Neuroanatomical studies on the mechanism of scalp acupuncture therapy using the pseudorabies virus (Pseudorabies virus(PRV)를 이용한 두침(頭針) 치료(治療) 기전(機轉)에 대한 신경해부학(神經解剖學的) 연구(硏究))

  • Lee, Tae-Young;Lee, Chang-Hyun;Lee, Sang-Ryoung;Yuk, Sang-Won;Lee, Kwang-Gyu;Yuk, Tae-Han
    • Journal of Acupuncture Research
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    • v.17 no.2
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    • pp.261-276
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    • 2000
  • 본 실험은 pseudorabies 바이러스 (PRV) 의 Bartha strain 을 안면신경의 측두지, 하지를 지배하는 신경 (좌골신경) 및 상지를 지배하는 신경 (요골, 척골, 정중신경) 에 주입한 후 4 일간의 생존시간이 경과한 후 척수와 뇌를 적출하여 동결절편을 제작한 후 면역조직화학적 염색기법과 X-gal 조직화학 염색법을 시행하여 염색된 신경세포체를 척수와 뇌에 투사된 공통영역을 관찰하고 두침의 영역중 하나인 운동구와 사지와의 관계에 대한 실험적 증거를 제시하고자 시행하였다. 위의 실험에서 얻어진 결과는 아래와 같다. 1. 안면신경의 측두지, 하지를 지배하는 신경 (좌골신경) 및 상지를 지배하는 신경 (요골, 측골, 정중신경) 에서 투사된 공통된 영역은 척수에서 경수의 층판 1-IV, 흉수의 intermediolateral nucleus(IML), dorsal nucleus(D) 및 층판 X, 요수의 층판 IV, V, 천수의 층판 IV, V, IX, X 등의 영역에서 관찰되었고, 뇌줄기에서는 caudoventrolateral reticular nucleus(CVL), nucleus solitary tract(Sol), rostroventrolateral nucleus(RVL), area postrema(AP), raphe nuclei(raphe pallidus, raphe obscurus, raphe magnus), inferior olivary nucleus 의 등쪽부분 (gigantocellular reticular nucleus, Gi), Kolliker-Fuse nucleus(KF), central gray(CG), dorsal raphe nucleus (DR) and A5 영역에 표지된다. 또한 paraventricular hypothalamic nucleus(PRV) 와 lateral hypothalamic reticular nucleus(LH)에서도 관찰되고 locus coeruleus(LC) 와 subcoeruleus nuc!eus(SubCA) 에서도 관찰된다.

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A Study on the Relationship among Job Attitude, Personality and Demographic Characteristics based on the Types of Dual Commitment (이중몰입유형별 직무태도와 성격 및 인구통계학적 특성의 융복합적 관계성 연구)

  • Kang, Young-Seok;Jeon, Sang-Gil;Lee, Mi-Kyeong
    • Journal of Digital Convergence
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    • v.14 no.7
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    • pp.135-144
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    • 2016
  • This study attempted to verify the relationship among Job Attitude, Personality and Demographic Characteristics according to Dual Commitment Type. The groups were named 'very high dual commitment group(both highly committed to the organization and union)', 'normal dual commitment group(average level of commitment to both of them)', 'resisting dual commitment group(no commitment to both of them)', 'organization commitment group(only committed to organization)', 'union commitment group(only committed to union)'. This research was analyzed by 388 Hyundai and Kia motor company members and 'very high dual commitment group' and 'organizational commitment group' showed the highest Job Satisfaction and Job Involvement. Through group segmentations considering sub-concepts could contribute to design a strategy to increase the company's competitiveness in aspects of the direction to the HR policy.

An Aerodynamic Study of Velopharyngeal Closure Function in Cleft Palate Patients (구개열 환자의 비인강폐쇄 기능에 대한 공기역학적 연구)

  • Ahn, Tae-Sub;Yang, Sang-Ill;Shin, Hyo-Keun
    • Speech Sciences
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    • v.1
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    • pp.237-259
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    • 1997
  • Cleft Palate speech appears to have hyper/hyponasality with velopharyngeal insufficiency and articulation disorders. Previous studies on Cleft Palate speech have shown that speech tends to have lower airflow and air pressure. To examine the aerodynamic characteristics of Cleft Palate speech, Aerophone II Voice function Analyzer was used. We measured sound pressure level, airflow, air pressure and glottal power. Three Cleft Palate adults and five normal adults participated in this experiment. The test words are composed of: (1) the sustained vowel /o/ (2) /CiCi/, where C is one of three different stop consonants in Korean (3) /bimi/. Subjects were asked to produce /bimi/ five times without opening their lips. All the data was statistically tested by t-test for Cleft Palate patients before operation groups and control groups and paired t-test for Cleft Palate patients before and after operation groups. The results were as follow: (1) Cleft Palate patients generally speak with incomplete oral closure and lower oral air pressure. As a result, the SPL of Cleft Palate before operation is 3 dB lower than control groups. (2) Airflow of Cleft Palate in phonation and articulation is lower than that of control groups. However, it increased after operation. Lung volume and mean airflow in phonation are significantly increased (p<0.05). (3) Although velopharyngeal function (velar opening rate) of Cleft Palate is poor in comparison with control groups, it was recovered after operation. In this event maximum flow rate and mean airflow rate are significantly increased (p<0.05). (4) Air pressure of Cleft Palate in speech is lower than that of control groups. In general, the air pressure of Cleft Palate increased after operation. In this event air pressure of glottalized consonant is significantly increased (p<0.04). (5) Glottal Power(mean power, mean efficient and mean resistant) of Cleft Palate patients is lower than that of control groups. But mean efficient and mean resistant of Cleft Palate patients increased significantly (p<0.05) after operation.

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Skin photoaging in reconstituted skin culture models (3D 피부세포 배양계를 이용한 피부광노화 연구)

  • 강상진
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.25 no.2
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    • pp.59-75
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    • 1999
  • Skin is continuously exposed to external stimuli including ultraviolet radiation, which is a major cause of skin photoaging. According to recent discoveries, UVA with a lower energy but deep-penetrating properties, compared to UVB, is likely to play a major part in causing skin photoaging. The clinical and histochemical changes of photoaging are well characterized, but the biochemical mechanisms are poorly understood partly due to the lack of suitable experimental systems. In this work, three-dimensional, reconstituted skin culture models were prepared. After certain period of maturation, the equivalent models were shown to be similar in structure and biochemical characteristics to normal skin. Mature dermal and skin equivalent models were exposed to sub-lethal doses of UVA, and the effects of UVA relevant to dermal photoaging were monitored, including the production of elastin, collagen, collagenase(MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1). Interestingly, dermal and skin equivalents reacted differently to acute and chronic exposure to UVA. Elastin production was increased as soon as one week after commencing UVA irradiation by chronic exposure, although a single exposure failed to do so. This early response could be an important advantage of equivalent models in studying elastosis in photoaged skin. Collagenase activity was increased by acute UVA irradiation, but returned to control levels after repeated exposure. On the other hand, collagen biosynthesis, which was increased by a single exposure, decreased slightly during 5 weeks of prolonged UVA exposure. Collagenase has been thought to be responsible for collagen degeneration in dermal photoaging. However, according to the results obtained in this study, elevated collagenase activity is not likely to be responsible for the degeneration of collagen in dermal photoagig, while reduced production of collagen may be the main reason. It can be concluded that reconstituted skin culture models can serve as useful experimental tools for the study of skin photoaging. These culture models are relatively simple to construct, easy to handle, and are reproducible Moreover the changes of dermal photoaging can be observed within 1-4 weeks of exposure to ultraviolet light compared to 4 months to 2 years for human or animal studies. These models will be useful for biochemical and mechanistic studies in a large number of fields including dermatology, toxicology, and pharmacology.

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PILOT INJECTION OF DME FOR IGNITION OF NATURAL GAS AT DUAL FUEL ENGINE-LIKE CONDITIONS

  • MORSY M. H.;AHN D. H.;CHUNG S. H.
    • International Journal of Automotive Technology
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    • v.7 no.1
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    • pp.1-7
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    • 2006
  • The ignition delay of a dual fuel system has been numerically investigated by adopting a constant volume chamber as a model problem simulating diesel engine relevant conditions. A detailed chemical kinetic mechanism, consisting of 28 species and 135 elementary reactions, of dimethyl ether (DME) with methane ($CH_{4}$) sub-mechanism has been used in conjunction with the multi-dimensional reactive flow KIVA-3V code to simulate the autoignition process. The start of ignition was defined as the moment when the maximum temperature in the combustion vessel reached to 1900 K with which a best agreement with existing experiment was achieved. Ignition delays of liquid DME injected into air at various high pressures and temperatures compared well with the existing experimental results in a combustion bomb. When a small quantity of liquid DME was injected into premixtures of $CH_{4}$/air, the ignition delay times of the dual fuel system are longer than that observed with DME only, especially at higher initial temperatures. The variation in the ignition delay between DME only and dual fuel case tend to be constant for lower initial temperatures. It was also found that the predicted values of the ignition delay in dual fuel operation are dependent on the concentration of the gaseous $CH_{4}$ in the chamber charge and less dependent on the injected mass of DME. Temperature and equivalence ratio contours of the combustion process showed that the ignition commonly starts in the boundary at which near stoichiometric mixtures could exists. Parametric studies are also conducted to show the effect of additive such as hydrogen peroxide in the ignition delay. Apart from accurate predictions of ignition delay, the coupling between multi-dimensional flow and multi-step chemistry is essential to reveal detailed features of the ignition process.