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Production of ColE1 Type Plasmid by Escherichia coli $DH5\alpha$ Cultured Under Nonselective Conditions

  • PASSARINHA L. A.;DIOGO M. M.;QUEIROZ J. A.;MONTEIRO G. A.;FONSECA L. P.;PRAZERES D. M. F.
    • Journal of Microbiology and Biotechnology
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    • v.16 no.1
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    • pp.20-24
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    • 2006
  • Plasmid DNA (pDNA) is a product of interest for many biopharmaceutical companies and research laboratories, because of increase in the number of gene therapy protocols that use nonviral vectors. This work was undertaken to study the effect of antibiotic and dissolved oxygen concentration (DOC) on the production of a ColE 1-type plasmid (pVAX1-LacZ) hosted in Escherichia coli $DH5\alpha$ and cultured in a batch fermentor with 0.751 of Terrific Broth. A decrease in the DOC from $60\%\;to\;5\%$ was shown to increase the specific pDNA concentration approximately 1.5-fold, due to the downregulation of growth. Additionally, this increase in the pDNA concentration led to a 2.2-fold increase in the purity of cell lysates obtained after cell lysis. However, the use of higher DOC led to 2.8-fold higher volumetric productivity as a consequence of a faster growth rate, reducing the fermentation time from 24 to 8 h. Interestingly, the specific pDNA concentration, and pDNA productivity and purity were always higher $(10-15\%)$ in the absence of antibiotic. Overall, the data indicate that nonselective conditions can be used without compromising yield, productivity, and purity of pDNA.

Validation of a Selective Method for Simultaneous Determination of Doxifluridine and 5-Fluorouracil in Dog Plasma by LC-MS/MS (LC/MS/MS를 이용한 비글견의 혈장 중 Doxifluridine 및 5-Fluorouracil의 동시 분석법 Validation)

  • Kim, Ghee-Hwan;Kim, Won;Kim, Jin-Sung;Jin, Qingri;Kang, Won-Ku;Lee, Jong-Hwa;Ha, Jung-Heun;Jeong, Eun-Ju
    • Journal of Pharmaceutical Investigation
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    • v.37 no.3
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    • pp.179-186
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    • 2007
  • A simple, sensitive and selective liquid chromatographic/tandem mass spectrometric method (LC-MS/MS) was developed and validated for doxifluridine and 5-fluorouracil (5-FU) quantification in dog heparinized plasma. Sample preparation was based on liquid-liquid extraction using a mixture of isopropanol/ethyl acetate (1/9 v/v) to extract doxifluridine, 5-FU and 5-chlorouracil (5-CU, an internal standard) from plasma. Chromatography was performed on a C-18 analytical column and the retention times were 2.7, 1.5 and 1.7 min for doxifluridine, 5-FU and 5-CU, respectively with shorter analysis time within 5 min than previously reported methods. The ionization was optimized using ESI negative mode and selectivity was achieved by tandem mass spectrometric analysis by multiple reaction monitoring (MRM) using the transformations of m/z 244.8>107.6, 129.0>42.0 and 144.9>42.1 for doxifluridine, 5-FU and 5-CU, respectively. The achieved low limit of quantification was 20.0 ng/mL and the assay exhibited linear range of 20-2000 ng/mL ($R^2>0.99957$ for doxifluridine and $R^2>0.99857$ for 5-FU), using $100{\mu}L$ of plasma. Accuracy and precision of quality control samples for both doxifluridine and 5-FU met KFDA and FDA Guidance criteria of 15% for accuracy with coefficients of variation less than 15%. This method demonstrated adequate sensitivity, specificity, accuracy, precision and stability to support the simultaneous analysis of doxifluridine and 5-FU in dog plasma samples in pharmacokinetic and bioequivalence studies.

Purification and Some Properties of Arginine Deiminase in Euglena gracilis Z (Euglena gracilis Z로부터 Arginine Deiminase의 정제 및 그의 특성)

  • Park, Bong-Sun;Hirotani, Aiko;Nakano, Yoshihisa;Kitaoka, Shozaburo
    • Journal of the Korea Organic Resources Recycling Association
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    • v.1 no.1
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    • pp.115-125
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    • 1993
  • In Euglena gracilis arginine deiminase was located in the mitochondrial matrix. The highly purified enzyme required $Co^{2+}$ for the enzyme reaction with the $K_m$ value of 0.23 nM, and its optimum pH was 9.7 to 10.3. The molecular weight of the native enzyme protein was 87,000 by gel filtration, and SDS-acrylamide gel electrophoresis showed that the enzyme consisted of two identical subunits with a molecular weight of 48,000. Euglena arginine deiminase was inhibited by sulfhydryl inhibitors, indicating that a sulfhydryl group is involved in the active center of the enzyme. It exhibited negative cooperativity in binding with arginine. $L-{\alpha}-amino-{\beta}-guanidino-propionate$, D-arginine, and L-homoarginine strongly inhibited the enzyme while ${\beta}-guanidinopro-pionate$, ${\gamma}-guanidinobutyrate$, and guanidinosuccinate did not. Considerable inhibition was also observed with citrulline and ornithine. We discuss the effects of the unique properties of the Euglena arginine deiminase on the regulation of arginine metabolism in this protozoon.

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Effect of Stepped Pattern of Feed Intake Using Rice Straw as Roughage Source on Regulation of Growth, Reproduction and Lactation in Dairy Heifers

  • Jin, M.G.;Lee, H.G.;Lee, H.J.;Hong, Z.S.;Wang, J.H.;Yin, Y.H.;Jin, R.H.;Cho, K.K.;Choi, Y.J.
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.6
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    • pp.794-798
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    • 2004
  • An attempt was made to improve the efficiency of growth and lactation performance of dairy heifers subjected to a stair-step growth scheme using rice straw as the sole roughage source. Twenty-four young Holstein heifers were randomly assigned to either control or test group. The control diet met the National Research Council (NRC) requirement, with heifers calving at 24 to 26 mo of age. The test group was individually fed according to a schedule of 3, 2, 4, 2, 5 and 2 mo in which feed intake was alternately 20% below or 25% above the NRC requirements. Heifers on the stair-step growth pattern gained more body weight and consumed less dry matter (10.80 and 11.22%, respectively), resulting an increasing growth efficiency compared with the control. Body condition, first estrus, first conception, services per conception and calving difficulty (data not shown) were not affected. Milk yield of the test group was 8.5% higher than that of the control group. During the early lactation period, the milk yield was significantly higher in the stair-step group than in that of the control group (p<0.05). Milk composition was not affected by compensatory growth induced by the stair-step scheme. Also, weight at calving and calf growth performance was not affected by stair-step growth. The results indicate that using rice straw as a sole roughage source in a stair-step compensatory growth scheme can contribute to the improvement of growth efficiency and early lactation performance.

Effect of Inhibitor of Glycogen Synthase Kinase 3 on Self-Renewal of Human Embryonic Stem Cells

  • Lee Eunyoung;Rho Jeung-yon;Yu Kwon;Paik Sang-Gi;Lee Kyung-Kwang;Han Yong-Mahn
    • Reproductive and Developmental Biology
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    • v.29 no.2
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    • pp.93-99
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    • 2005
  • Human embryonic stem cells (hESCs) derived from the inner cell mass of blastocysts have the ability to renew themselves and to differentiate into cell types of all lineage. The present study was carried out to investigate whether the Wnt signaling pathway is related to maintaining self-renewal of hESCs. Glycogen Synthase Kinase 3 (GSK-3) inhibitor, BIO ((2'Z,3'E)-6-Bromoindirubin-3'-oxime) was treated to Miz-hES1 line for activation of Wnt signaling pathway. BIO-nontreated hESCs (control) and BID-treated hESCs were cultured for 5 days in the modified feeder-free system. During the culture of hESCs, differences were observed in the colony morphology between 2 groups. Controls were spread outwards whereas BIO-nontreated hESCs were clumped in the center and the differentiated cells were spreading outwards in the edges. The results of stem cell specific marker staining indicated that control were differentiated in large part whereas BIO-treated hESCs maintain self-renewal in the center of the colony. The results of lineage marker staining suggested that outer cells of the hESC colony were differentiated to the neuronal progenitor cells in both control and BIO-treated hESC. These results indicate that Wnt signaling is related to self-renewal in hESCs. In addition, control group showed higher composition of apoptotic cells $(23.76\%)$ than the BID-treated group $(5.59\%)$. These results indicate that BIO is effective on antapoptosis of hESCs.

Comparison of Motility, Acrosome, Viability and ATP of Boar Sperm with or without Cold Shock Resistance in Liquid Semen at 17℃ and 4℃, and Frozen-thawed Semen

  • Yi, Y.J.;Li, Z.H.;Kim, E.S.;Song, E.S.;Kim, H.B.;Cong, P.Q.;Lee, J.M.;Park, Chang-Sik
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.2
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    • pp.190-197
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    • 2008
  • This study was designed to analyze boar sperm to compare motility, acrosome morphology, viability and ATP by various preservation methods between Duroc boar A with cold shock resistance sperm and Duroc boar B with cold shock sensitivity sperm. Semen volume, sperm concentration, motility and normal acrosome between Duroc boar A and B did not show any differences within 2 h after collection. There were no differences in sperm motility and normal acrosome between boar A and B at 1 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively. However, sperm motility and normal acrosome from 2 day of preservation at $17^{\circ}C$ and $4^{\circ}C$, respectively, were higher for boar A than boar B. The frozen-thawed sperm motility and normal acrosome were higher for boar A than boar B. The sperm viability and ATP concentration according to storage period of liquid semen at $17^{\circ}C$ and $4^{\circ}C$ were higher for boar A than boar B. Also, the sperm viability and ATP concentration of frozen-thawed semen were higher for boar A than boar B. In conclusion, we found out that the original quality of boar semen with cold shock resistance sperm played an important role.

Quality Characteristics of Korean Malt Barley Varieties (한국산(韓國産) 맥주맥(麥酒麥)의 품질특성(品質特性)에 관(關)한 연구(硏究))

  • Shin, H.K.;Kim, Y.S.;Bae, S.H.;Kim, Z.U.
    • Applied Biological Chemistry
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    • v.23 no.3
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    • pp.150-156
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    • 1980
  • Local malt barleys have been examined for the quality characteristics. Regional differences in quality characteristics of malt barley were observed in a single variety. Promising barley varieties for brewing, in terms of protein contents, germinating power and diastic power were Sacheon No. 2,6,9, Mokpo 11 and 12. Possible use of certain malt barleys is suggested and discussed.

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Severity-Adjusted Mortality Rates of Coronary Artery Bypass Graft Surgery Using MedisGroups (MedisGroups를 이용한 관상동맥우회술의 중증도 보정사망률에 관한 연구)

  • Kwon, Young-Dae
    • Quality Improvement in Health Care
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    • v.7 no.2
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    • pp.218-228
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    • 2000
  • Background : Among 'structure', 'process' and 'outcome' approaches, outcome evaluation is considered as the most direct and best approach to assess the quality of health care providers. Risk-adjustment is an essential method to compare outcome across providers. This study has aims to judge performance of hospitals by severity adjusted mortality rates of coronary artery bypass graft (CABG) surgery. Methods : Medical records of 584 patients who got the CABG surgery in 6 general hospitals during 1996 and 1997 were reviewed by trained nurses. The MedisGroups was used to quantify severity of patients. The predictive probability of death was calculated for each patient in the sample from a multivariate logistic regression model including the severity score, age and sex. For evaluation of hospital performance, we calculated ratio of observed number to expected number of deaths and z score [(observed number of deaths - expected number of deaths)/square root of the variance in the number of deaths], and compared observed mortality rate with confidence interval of adjusted mortality rate for each hospital. Results : The overall in-hospital mortality was 7.0%, ranged from 2.7% to 15.7% by hospital. After severity adjustment the mortality by hospital was from 2.7% to 10.7%. One hospital with poor performance was distinctly divided from others with good performance. Conclusion : In conclusion, severity-adjusted mortality rate of CABG surgery might be applied as an indicator for hospital performance evaluation in Korea. But more pilot studies and improvement of methodologies has to be done to use it as quality indicator.

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ON INJECTIVITY AND P-INJECTIVITY, IV

  • Chi Ming, Roger Yue
    • Bulletin of the Korean Mathematical Society
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    • v.40 no.2
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    • pp.223-234
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    • 2003
  • This note contains the following results for a ring A : (1) A is simple Artinian if and only if A is a prime right YJ-injective, right and left V-ring with a maximal right annihilator ; (2) if A is a left quasi-duo ring with Jacobson radical J such that $_{A}$A/J is p-injective, then the ring A/J is strongly regular ; (3) A is von Neumann regular with non-zero socle if and only if A is a left p.p.ring containing a finitely generated p-injective maximal left ideal satisfying the following condition : if e is an idempotent in A, then eA is a minimal right ideal if and only if Ae is a minimal left ideal ; (4) If A is left non-singular, left YJ-injective such that each maximal left ideal of A is either injective or a two-sided ideal of A, then A is either left self-injective regular or strongly regular : (5) A is left continuous regular if and only if A is right p-injective such that for every cyclic left A-module M, $_{A}$M/Z(M) is projective. ((5) remains valid if 《continuous》 is replaced by 《self-injective》 and 《cyclic》 is replaced by 《finitely generated》. Finally, we have the following two equivalent properties for A to be von Neumann regula. : (a) A is left non-singular such that every finitely generated left ideal is the left annihilator of an element of A and every principal right ideal of A is the right annihilator of an element of A ; (b) Change 《left non-singular》 into 《right non-singular》in (a).(a).

Effect of Grain Angle on Bending Properties of Pinus densiflora (소나무재의 휨 가공성에 미치는 섬유경사각의 영향)

  • Kim, Jung-Hwan;Lee, Weon-Hee
    • Journal of the Korean Wood Science and Technology
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    • v.29 no.2
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    • pp.118-125
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    • 2001
  • In this study, it was examined the characteristics of bending property of red pine(Pinus densiflora S, et Z.) related to slope of grain. At first, we have investigated the characteristics of wood species for bending property. At second, it was examined the relationships between grain angle and its related bending property. Specimens were made following to grain angle $0^{\circ}$, $30^{\circ}$, $45^{\circ}$, $60^{\circ}$, $90^{\circ}$, respectively. Dimension of wood materials was $10mm(T){\times}20mm(R){\times}350mm(L)$. Microwave irradiation time for bending process was 30, 60, 90, 120 seconds. The result of this study were as follows ; 1. Grain angle of wood was closely related to Young's modulus on bending process. In the process of bending with various grain angle, wood bending was easily proceed on the high grain angle range. 2. However, the strength of bent wood was very weak when the grain angle was high. Therefore, it was considered suitable grain angle for bending was existed. 3. The characteristics of wood properties for wood bending were very different among wood species.

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