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Enzymatic Manufacture of Deoxythymidine-5'-Triphosphate with Permeable Intact Cells of E. coli Coexpressing Thymidylate Kinase and Acetate Kinase

  • Zhang, Jiao;Qian, Yahui;Ding, Qingbao;Ou, Ling
    • Journal of Microbiology and Biotechnology
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    • v.25 no.12
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    • pp.2034-2042
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    • 2015
  • A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25℃ for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'-triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

The Effects of Glucose, Insulin and Angiotensin II on Plasminogen Activator Inhibitor-1 Expression and Growth of Aortic Vascular Smooth Muscle Cell in Rats (포도당, 인슐린 및 Angiotensin II가 흰쥐 대동맥평활근세포의 Plasminogen Activator Inhibitor-1 발현 및 성장에 미치는 영향)

  • 최세영;이인규;한승세;김재현;박창권;이광숙;유영선;김기식;김윤년
    • Journal of Chest Surgery
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    • v.32 no.4
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    • pp.333-340
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    • 1999
  • Background: Plasminogen activator inhibitor-1(PAI-1) is known as the primary physiological inhibitor of tissue-type plasminogen activator(t-PA) in the plasma, and is present within the atherosclerotic vessels. Increased plasma levels of PAI-1 are one of the major disturbances of the hemostatic system in patients with diabetes and/or hypertension, and may have multiple interrelations with the important risk factors in the development of atherosclerosis. This study was performed to determine whether altered gene expression of PAI-1 occurs within the arterial wall, and thereby potentially contributing to the increase of cardiovascular risks associated with diabetes and/or hypertension. Material and Method: The aortic vascular smooth muscle cells of the rat were exposed to 22 mM glucose, angiotensin II, and insulin increased PAI-1 mRNA expression with the use of Northern blotting were examined. Also examined were the effects of 22 mM glucose, angiotensin II and insulin on the growth of the rat's aortic smooth muscle cells by using MTT assay. Result: Twenty-two mM glucose treatment increased the PAI-1 mRNA expression in a time- and dose-dependent manner. Aniotensin II treatment synergistically increased the glucose-induced PAI-1 mRNA expression. In contrast, addition of insulin attenuated the increase of 22 mM glucose and angiotensin II induced PAI-1 mRNA expression. Furthermore, treatment of 22 mM glucose, angiotensin II and insulin resulted in a significant increase in cell numbers. This study demonstrated that 22 mM glucose and angiotensin II have a synergistic effect in stimulating the PAI-1 mRNA expression and in the cell growth of the rat's aortic smooth muscle cells. Conclusion: Elevation of glucose and angiotensin II may be important risk factors in impairing fibrinolysis and developing atherosclerosis in diabetic patients.

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The Effects of Ammonium Citrate and Ammonium Succinate on the Growth of Cells and Nitrogen Absorption in Korean Ginseng Suspension Cultures (고려인삼(Panax ginseng C.A. Meyer) 배양세포의 생육 및 질소 흡수에 미치는 Ammonium Citrate와 Ammonium Succinate의 영향)

  • 김홍성;김명원;소상섭;강영희
    • Journal of Plant Biology
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    • v.24 no.1
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    • pp.13-19
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    • 1981
  • In order to investigate the effects of ammonium citrate and ammonium succinate on the growth and absorption of nitrogen compounds supplied in the medium, Korean ginseng (Panax ginseng C. A. Meyer) calli were suspension cultured in MS medium with various concentrations of ammonium citrate and ammonium succinate. When Korean ginseng calli were cultured with 10 mM ammonium citrate, 10 mM ammonium succinate, and 10 mM ammonium nitrate (control) in MS media as the nitrogen sources, the growth, $NO_3$-N absorption and total nitrogen content of the Korean ginseng cells were greatest in the ammonium citrate and ammonium succinate concentrations. When Korean ginseng calli were cultured with 5 mM ammonium citrate and 5 mM ammonium succinate, the growth and nitrogen content were superior to those of the control: however, $NO_3$-N and $NH_4$-N absorptions were similar to those of the control. In conclusion, the 10 mM ammonium citrate and 10 mM ammonium succinate may be better able to facilitate the growth and $NO_3$

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Growth, Flowering, and Ripening Seed Characteristics of Mammillaria goldii, M. theresae, and M. pseudopectinata (선인장 Mammillaria goldii, M. theresae와 M. pseudopectinata의 생육, 개화 및 종자형성 특성)

  • Song, Cheon Young;Lee, Dong Chul;Park, In Tae;Park, Wha Soon;Hong, Seung Min
    • FLOWER RESEARCH JOURNAL
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    • v.17 no.4
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    • pp.285-290
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    • 2009
  • This experiment was conducted to investigate the propagation system by seed to check growth, flowering, and making seed of three-year-old cactus of potted Mammillaria goldii. M. theresae, and M. pseudopeclinata. Plant height and width of them was 2.5~2.8 cm, and 2.8~3.1 cm, and seed formation of Mammillaria goldii and M. theresae had internal body taking130 to 135 days to ripening seeds, however, M. pseudopectinata had externals taking just 26 days to ripening seeds. The mean seeded pod from April to June of M. goldii was 50.0% and the number of seeds in a pod was 25.3. In M. theresae, the mean seeded pod from April to June was 57.3% and the number of seeds in a pod was 35.0. However, the highest rate of seeded pod on May of M. pseudopectinata was 75.0% and the number of seed in a pod was 66.0. The sib crossing was much more ripening seed fertilization than that of self cross ing in the Mammillaria. The self crossing was formed from 12.0% to 20.0% of seeded pod, from 12.0 to 16.0 number of seed in a pod. However, sib crossing obtained 58.0% of seeded pod, 30 seeds in a pod of M. goldii, 65.0% seeded pods and 40 seeds in a pod of M. theresae, and 75.0% seeded pod, 68 seeds in a pod of M. pseudopectinala as the highest of them.

Studies on Hematologic Values and Blood Chemistry Values of Normal Jindo Dogs 3. Serum Electrolyte Values for Adult Jindo Dogs (진도견(珍島犬)의 혈액상(血液像) 및 혈액화학치(血液化學値)에 관(關)한 연구(硏究) 제삼보(第三報) 진도견(珍島犬)의 혈청전해질치(血淸電解質値))

  • Park, Nam-Yong;Rim, Bong-Ho;Cho, Sung-Man
    • Korean Journal of Veterinary Research
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    • v.20 no.2
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    • pp.175-178
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    • 1980
  • Normal values of serum electrolytes were determined for adult 36 females and 18 males, 1 to 6 years old, of Jindo dogs. The mean values, standard deviation and rangEs of sodium, potassium, chloride, calcium and inorganic phosphorus were studied and data analyzed to determine whether differences existed between the values of female and those of male dogs. The results obtained in this study were as follows: 1. The mean value of serum sodium was $143{\pm}1.5mEq/liter$, ranged from 138mEq/liter to 152 mEq/liter. The mean values for the females and males were $144{\pm}2.0mEq/liter$ and $142{\pm}1.7mEq/liter$, respectively. They showed that the female dogs had a tendency toward higher values than the male dogs, but no significant sex-related differences were observed. 2. The mean value of serum potassium was $4.6{\pm}0.2mEq/liter$, ranged frem 3.5mEq/liter to 6.0mEq/liter. The mean values for the females and males were $4.7{\pm}0.4mEq/liter$ and $4.5{\pm}0.3mEq/liter$. They showed that the female dogs had a tendency toward higher values than the male dogs, but sex differences were not found to be significant. 3. The mean value of serum chloride was $103{\pm}3.0mEq/liter$ ranged from 95mEq/liter to 117mEq/liter. The mean values for the females and males were $103{\pm}3.2mEq/liter$ and $103{\pm}3.3mEq/liter$, no sex differences showed. 4. The mean value of serum calcium was $5.2{\pm}0.1mEq/liter$ ranged from 4.7mEq/liter to 5.8mEq/liter. The mean values for the females and males were $5.4{\pm}0.1mEq/liter$ and $5.2{\pm}0.09mEq/liter$ respectively, no significant sexd iffere-nces were observed. 5. The mean value of serum inorganic phosphorus was $4.3{\pm}0.7mg/100ml$, ranged from 2.5mg/100ml to 5.8mg/100ml. The mean values for the females and males were $4.3{\pm}0.9mg/100ml$ and $4.1{\pm}0.8mg/100ml$, respectively, no sex differences showed.

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Identification of Glomerella cingulata from Dracaena sanderiana (Dracaena sanderiana로부터 Glomerella cingulata의 동정)

  • Seo, Il-Gyo;Shim, Chang-Ki;Kim, Dong-Gil;Bae, Dong-Won;Kim, Hee-Kyu
    • Research in Plant Disease
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    • v.8 no.4
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    • pp.224-227
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    • 2002
  • From the necrotic stem of aquaculture Dracaena for living room decoration, we observed conidia on acervuli with occasional setae and perithecia in vivo. Morphological characters were; acervuli, dark brown and 300-500 ${\mu}{\textrm}{m}$ in diameter; conidia, hyaline, aseptate, cylindrical to slightly ellipsoid with rounded apex and narrow truncate base on phialidic conidiophores, 12.5-17.5 ${\mu}{\textrm}{m}$; setae, variable in length 1-4 septate, slightly swollen at the base and tapered to the apex, 200 ${\mu}{\textrm}{m}$; appressoria, dark brown ovate to obovate irregular 5-12.5 ${\mu}{\textrm}{m}$, perithecia on decayed stem, globose, dark brown and black 85-300 ${\mu}{\textrm}{m}$; asci 8 spored, clavate to cylindrical; 50-62.5$\times$8-10 ${\mu}{\textrm}{m}$; ascospores, oval to fusiform, sometimes slightly curved, aseptate, hyaline. Above characteristics were also confirmed in vitro. Colletotrichum state of this fungus was also compared with C. lindemuthianum and C. musae in terms of morphology of conidia and setae and growth on PDA etc. This fungus was identified as Glomerella cingulata(Stonem.) Spaulding & Schrenk(Colletotrichum gloeosporioides(Penz. Sacc.).

Measurement of Clothing Pressure of Bell-Bottom Slcaks according to Movements of Legs (벨버텀 슬랙스(Bell-Bottom Slacks)의 동작에 따른 하지 부위별 피복압의 측정)

  • 권윤희;이연순;박세진
    • Proceedings of the ESK Conference
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    • 1997.04a
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    • pp.256-262
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    • 1997
  • The purpose of this study was to investigate the effects of clothing pressure of Bell-bottom slacks according to various movements of the legs In this study, movements of legs were classified by M1, M2, M3, M4, M5, M6. (M1: erecting Position, M2: Setting Position, M3: Stepping Pssition, M4: Leapfrogging Position, M5: Sit-on-one's Position, M6: Traditional noble-sitting) The results were as follows: clothing pressure was very different according to the movements of the legs and was in order M4>M5>M6>M2>M3>M1. Particually, clothing pressure in the knee point is the highest in the M4 movement $(550.81g/cm^{2})$.

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Fracture toughness of high performance concrete subjected to elevated temperatures Part 2 The effects of heating rate, exposure time and cooling rate

  • Zhang, Binsheng;Cullen, Martin;Kilpatrick, Tony
    • Advances in concrete construction
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    • v.5 no.5
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    • pp.513-537
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    • 2017
  • In this study, the fracture toughness $K_{IC}$ of high performance concrete (HPC) was investigated by conducting three-point bending tests on a total of 240 notched beams of $500mm{\times}100mm{\times}100mm$ subjected to heating temperatures up to $450^{\circ}C$ with exposure times up to 16 hours and various heating and cooling rates. For a heating rate of $3^{\circ}C/min$, $K_{IC}$ for the hot concrete sustained a monotonic decrease trend with the increasing heating temperature and exposure time, from $1.389MN/m^{1.5}$ at room temperature to $0.942MN/m^{1.5}$ at $450^{\circ}C$ for 4-hour exposure time, $0.906MN/m^{1.5}$ for 8-hour exposure time and $0.866MN/m^{1.5}$ for 16-hour exposure time. For the cold concrete, $K_{IC}$ sustained a two-stage decrease trend, dropping slowly with the heating temperature up to $150^{\circ}C$ and then rapidly down to $0.869MN/m^{1.5}$ at $450^{\circ}C$ for 4-hour exposure time, $0.812MN/m^{1.5}$ for 8-hour exposure time and $0.771MN/m^{1.5}$ for 16-hour exposure time. In general, the $K_{IC}$ values for the hot concrete up to $200^{\circ}C$ were larger than those for the cold concrete, and an inverse trend was observed thereafter. The increase in heating rate slightly decreased $K_{IC}$, and at $450^{\circ}C$ $K_{IC}$ decreased from $0.893MN/m^{1.5}$ for $1^{\circ}C/min$ to $0.839MN/m^{1.5}$ for $10^{\circ}C/min$ for the hot concrete and from $0.792MN/m^{1.5}$ for $1^{\circ}C/min$ to $0.743MN/m^{1.5}$ for $10^{\circ}C/min$ for the cold concrete after an exposure time of 16 hours. The increase in cooling rate also slightly decreased $K_{IC}$, and at $450^{\circ}C$ $K_{IC}$ decreased from $0.771MN/m^{1.5}$ for slow cooling to $0.739MN/m^{1.5}$ for fast cooling after an exposure time of 16 hours. The fracture energy-based fracture toughness $K_{IC}$' was also assessed, and similar decrease trends with the heating temperature and exposure time existed for both hot and cold concretes. The relationships of two fracture toughness parameters with the weight loss and the modulus of rapture were also evaluated.

Effects of High Glucose Levels on the Protein Kinase C Signal Transduction Pathway in Primary Cultured Renal Proximal Tubule Cells

  • Han, Ho-Jae;Kang, Ju-Won;Park, Kwon-Moo
    • The Korean Journal of Physiology
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    • v.30 no.2
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    • pp.257-267
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    • 1996
  • Diabetes mellitus is associated with a wide range of pathophysiologic changes in the kidney. This study was designed to examine the mechanisms by which glucose modulates the expression of polarized membrane transport functions in primary cultured rabbit renal proximal tubule cells. Results are as follows: The rate of 30 minute $Rb^{+}$ uptake was significantly higher($137.76{\pm}5.40%$) in primary renal tubular cell cultures treated with 20 mM glucose than that of 5 mM glucose. Not the level of mRNA for the ${\alpha}$ subunit of Na, K-ATPase but that of ${\beta}$ subunit was elevated in primary cultures treated with high glucose. The initial rate of methyl-${\alpha}$-D-glucopyranoside(${\alpha}$-MG) uptake was significantly lower($71.91{\pm}3.02%$) in monolayers treated with 20 mM glucose than that of 5 mM glucose. There was a tendency of an increase in phlorizin binding site in cells treated with 5 mM glucose. However, 3-O-methyl-D-glucose(3-O-MG) uptake was not affected by glucose concentration in culture media. TPA inhibited $Rb^{+}$ uptake by $63.61{\pm}1.94\;and\;45.80{\pm}1.36%$ and ${\alpha}$-MG uptake by $48.54{\pm}3.69\;and\;41.87{\pm}6.70%$ in the cells treated with 5 and 20 mM glucose, respectively. Also TPA inhibited mRNA expression of Na/glucose cotransporter in cells grown in 5mM glucose medium. cAMP significantly stimulated ${\alpha}$-MG uptake by $114.65{\pm}5.70%$ in cells treated with 5mM glucose, while it did not affect ${\alpha}$-MG uptake in cell treated with 20 mM glucose. However, cAMP inhibited $Rb^{+}$ uptake by $76.69{\pm}4.16\;and\;66.87{\pm}2.41%$ in cells treated with 5 and 20 mM glucose, respectively. In conclusion, the activity of the renal proximal tubular Na,K-ATPase is elevated in high glucose concentration. In contrast, the activity of the Na/glucose cotransport system is inhibited. High glucose may in part affect the activity of the Na,K-ATPase and the Na/glucose cotransport system by controlling the protein kinase C and/or A signal transduction pathway in primary cultured renal proximal tubule cells.

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SURFACE ROUGHNESS OF COMPOSITE RESIN ACCORDING TO FINISHING METHODS (복합레진 표면의 연마방법에 따른 표면조도)

  • Min, Jeong-Bum;Cho, Kong-Chul;Cho, Young-Gon
    • Restorative Dentistry and Endodontics
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    • v.32 no.2
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    • pp.138-150
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    • 2007
  • The purpose of this study was to evaluate the difference of surface roughness of composite resin according to composite resin type, polishing methods, and use of resin sealant. Two hundred rectangular specimens, sized $8{\times}3{\times}2mm$, were made of Micro-new (Bisco, Inc., Schaumburg, IL, U.S.A) and Filtek Supreme (3M ESPE Dental Products, St. Paul, MN, U.S.A.), and divided into two groups; Micronew-M group, Filtek Supreme-S group. Specimens for each composite group were subdivided into five groups by finishing and polishing instruments used; M1 & S1(polyester strip), M2 & S2 (Sof-Lex disc), M3 & S3 (Enhance disc and polishing paste), M4 & S4(Astropol) and M5 & S5 (finishing bur), Polished groups were added letter B after the application of resin surface sealant (Biscover), eg, M1B and S1B. After specimens were stored with distilled water for 24hr, average surface roughness (Ra) was taken using a surface roughness tester. Representative specimens of each group were examined by FE-SEM (S-4700: Hitachi High Technologies Co., Tokyo, Japan). The data were analysed using paired t-test, ANOVA and Duncan's tests at the 0.05 probability level. The results of this study were as follows ; 1. The lowest Ra was achieved in all groups using polyester strip and the highest Ra was achieved in M5, S5 and M5B groups using finishing bur. On FE-SEM, M1 and S1 groups provided the smoothest surfaces, M5 and S5 groups were presented the roughest surfaces and voids by debonding of filler on the polished specimens. 2. There was no significant difference in Ra between Micronew and Filtek Supreme before the application of resin sealant, but Micronew was smoother than Filek Supreme after the application of resin sealant. 3. There was significant corelation between Ra of type of composite resin and polishing methods before the application of resin sealant (p=0.000), but no significant interaction between them after the application of resin sealant. On FE-SEM, most of composite resin surfaces were smooth after the application of resin sealant on the polished specimens. 4. Compared with before and after the application of resin sealant in group treated in the same composite and polishing methods, Ra of M4B and M5B was statistically lower than that of M4 and M5, and S5B was lower than that of S5, respectively (p<0.05). In conclusion, surface roughness by polishing instruments was different according to type of composite resin. Overall, polyester strip produced the smoothest surface, but finishing bur produced the roughest surface. Application of resin sealant provided the smooth surfaces in specimens polished with Enhance, Astropol and finishing bur, but not provided them in specimens polished with Sof-Lex disc.