• Title/Summary/Keyword: Cell culture condition

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Optimization of Culture Conditions for 1,3-propanediol Production from Glycerol Using Klebsiella pneumoniae (글리세롤로부터 1,3-propanediol 생산을 위한 Klebsiella pneumoniae 배양 조건 최적화)

  • Jun, Sun-Ae;Kong, Sean W;Sang, Byoung-In;Um, Youngsoon
    • Korean Chemical Engineering Research
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    • v.47 no.6
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    • pp.768-774
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    • 2009
  • To improve the productivity of 1,3-propanediol(1,3-PD) with K. pneumoniae DSM4799 using pure glycerol and crude glycerol derived from the biodiesel process, optimizing fermentation conditions was performed by changing environmental factors such as anaerobic/aerobic condition, temperature, glycerol concentration, and pH. When anaerobic conditions were maintained, there was an improved 1,3-PD production compared with that from aerobic/anaerobic 2-stage fermentation. From the results with temperature $26{\sim}37^{\circ}C$, the higher 1,3-PD production yield was observed at $30{\sim}33^{\circ}C$. For an initial glycerol concentration higher than 60 g/L, cell growth and 1,3-PD production were inhibited. When crude glycerol was used, the initial 1,3-PD production appeared to be inhibited. After 48 hr of incubation, however, 1,3-PD production with crude glycerol was even higher than that with pure glycerol, demonstrating the feasibility of 1,3-PD production using crude glycerol as a substrate. Fed-batch fermentation was applied for the high concentration of 1,3-PD without substrate inhibition. By regulating pH at 7 during the fed-batch with glycerol lower than 40 g/L, the yield of 1,3-PD was 25% higher than that without pH regulation(0.56 g/g vs. 0.45 g/g). In conclusion, based on our results, anaerobic conditions, temperature at $30^{\circ}C$, pure or crude glycerol lower than 40 g/L, and pH regulation at 7 were the optimized conditions for 1,3-PD production using K. pneumoniae DSM4799, making it more feasible to produce 1,3-PD at higher concentration and a lower price.

Introduction and Expression of PAP gene using Agrobacterium in Scrophularia buergeriana Miquel (Agrobacterium을 이용한 PAP 유전자의 현삼으로 도입 및 형질발현)

  • Yu, Chang-Yeon;Seong, Eun-Soo;Lim, Jung-Dae;Huang, Shan-Ai;Chae, Young-Am
    • Korean Journal of Medicinal Crop Science
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    • v.9 no.2
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    • pp.156-165
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    • 2001
  • Exogeneous application of pokeweed antiviral protein (PAP), a ribosomal-inacivating protein in the cell wall of Phytolacca americana (pokeweed) protects heterologous plants from viral and fungal infection. A cDNA clone of PAP introduced into Scrophularia buergeriana Miquel by thransformation with Agrobacterium tumefaciences. For plant transformation, explants were precultured on shoot induction medium without kanamycin for 2-5 day, and then they were cocultured with Agrobacterium for 10 minutes. The explants were placed on co culture medium in dark condition, $28^{\circ}C$ for 2days. After explants were washed in MS liquid medium, they were transferred into selection medium including kanamycin 50mg/L (MS salts+1mg/ l BAP+2mg/ l TDZ+0,2mg/ l NAA+MS vitamin+3% sucrose+0.8% agar, pH5.8). From PCR analysis, NPT II band was confirmed in transgenic plant genome and showed resistance against fungi in antifungal activity test. Micro assay to which protein extracted from transgenic line were added, revealed hyphae growth inhibition and no spore germination at high concentration. The characteristics of inhibited hyphae was represented transparent and thin. Expression of PAP in transgenic plants offers the possibility of developing resistance to viral and fungal infection.

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A COMPARATIVE STUDY OF PRESERVING ABILITY OF HUMAN PERIODONTAL LIGAMENT CELLS STORED IN DIFFERENT TEMPERATURED STORAGE MEDI (저장용액의 온도에 따른 치주인대세포의 생존율)

  • Jo, Jae-Hyun;Kim, Seong-Oh;Choi, Hyung-Jun;Lee, Jae-Ho;Son, Heung-Kyu;Choi, Byung-Jai
    • Journal of the korean academy of Pediatric Dentistry
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    • v.34 no.1
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    • pp.36-42
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    • 2007
  • To compare the survival rate of periodontal ligament cells preserved in storage media with good availability at the time of an avulsion injury, periodontal ligament cells were incubated in ${\alpha}-MEM$ culture medium containing 10% FBS in condition of $37^{\circ}C$, 5% $CO_2$. These cells were then cultured in HBSS, ${\alpha}-MEM$, milk(S co., P. co.) and tap water at the temperature of 4, 25, $37^{\circ}C$ each in 60 min. The groups were measured by MTT assay. The results were as follows : 1. Among the storage media at $4^{\circ}C$, ${\alpha}-MEM$ and P-milk had the highest preserving ability of periodontal ligament cells, while that of HBSS S-milk and tap was low in order. 2. Among the storage media at $25^{\circ}C$, ${\alpha}-MEM$ had the highest preserving ability of periodontal ligament cells, while that of P-milk, HBSS, S-milk, tap water was low in order. 3. Among the storage media at $37^{\circ}C$, the preserving ability of periodontal ligament cells was very high in ${\alpha}-MEM$, P-milk, HBSS and S-milk, it's lowest in tap water. 4. The preserving ability of periodontal ligament cells in ${\alpha}-MEM$ was high at $4^{\circ}C$ and it's low in order of $25^{\circ}C$, $37^{\circ}C$, but in HBSS was high at $4^{\circ}C$ and it's low at $25^{\circ}C$, $37^{\circ}C$ 5. The preserving ability of periodontal ligament cells in S-milk and P-milk was high at $4^{\circ}C$, $25^{\circ}C$ and it s low at $37^{\circ}C$. In conclusion, HBSS is the storage medium of choice in an avulsion, but in this study it is preferable to choose milk at $4^{\circ}C$ for tooth since it is easy to get and affect cell viability.

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The Effect of Nitric Oxide Donor or Nitric Oxide Synthase Inhibitor on Oxidant Injury to Cultured Rat Lung Microvascular Endothelial Cells (산화질소 공여물과 산화질소 합성효소 길항제가 백서 폐미세혈관 내피세포 산화제 손상에 미치는 영향)

  • Chang, Joon;Michael, John R.;Kim, Se-Kyu;Kim, Sung-Kyu;Lee, Won-Young;Kang, Kyung-Ho;Yoo, Se-Hwa;Chae, Yang-Seok
    • Tuberculosis and Respiratory Diseases
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    • v.45 no.6
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    • pp.1265-1276
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    • 1998
  • Background : Nitric oxide(NO) is an endogenously produced free radical that plays an important role in regulating vascular tone, inhibition of platelet aggregation and white blood cell adhesion to endothelial cells, and host defense against infection. The highly reactive nature of NO with oxygen radicals suggests that it may either promote or reduce oxidant-induced cell injury in several biological pathways. Oxidant injury and interactions between pulmonary vascular endothelium and leukocytes are important in the pathogenesis of acute lung injury, including acute respiratory distress syndrome(ARDS). In ARDS, therapeutic administration of NO is a clinical condition providing exogenous NO in oxidant-induced endothelial injury. The role of exogenous NO from NO donor or the suppression of endogenous NO production was evaluated in oxidant-induced endothelial injury. Method : The oxidant injury in cultured rat lung microvascular endothelial cells(RLMVC) was induced by hydrogen peroxide generated from glucose oxidase(GO). Cell injury was evaluated by $^{51}$chromium($^{51}Cr$) release technique. NO donor, such as S-nitroso-N-acetylpenicillamine(SNAP) or sodium nitroprusside(SNP), was added to the endothelial cells as a source of exogenous NO. Endogenous production of NO was suppressed with N-monomethyl-L-arginine(L-NMMA) which is an NO synthase inhibitor. L-NMMA was also used in increased endogenous NO production induced by combined stimulation with interferon-$\gamma$(INF-$\gamma$), tumor necrosis factor-$\alpha$(TNF-$\alpha$), and lipopolysaccharide(LPS). NO generation from NO donor or from the endothelial cells was evaluated by measuring nitrite concentration. Result : $^{51}Cr$ release was $8.7{\pm}0.5%$ in GO 5 mU/ml, $14.4{\pm}2.9%$ in GO 10 mU/ml, $32.3{\pm}2.9%$ in GO 15 mU/ml, $55.5{\pm}0.3%$ in GO 20 mU/ml and $67.8{\pm}0.9%$ in GO 30 mU/ml ; it was significantly increased in GO 15 mU/ml or higher concentrations when compared with $9.6{\pm}0.7%$ in control(p < 0.05; n=6). L-NMMA(0.5 mM) did not affect the $^{51}Cr$ release by GO. Nitrite concentration was increased to $3.9{\pm}0.3\;{\mu}M$ in culture media of RLMVC treated with INF-$\gamma$ (500 U/ml), TNF-$\alpha$(150 U/ml) and LPS($1\;{\mu}g/ml$) for 24 hours ; it was significantly suppressed by the addition of L-NMMA. The presence of L-NMMA did not affect $^{51}Cr$ release induced by GO in RLMVC pretreated with INF-$\gamma$, TNF-$\alpha$ and LPS. The increase of $^{51}Cr$ release with GO(20 mU/ml) was prevented completely by adding 100 ${\mu}M$ SNAP. But the add of SNP, potassium ferrocyanate or potassium ferricyanate did not protect the oxidant injury. Nitrite accumulation was $23{\pm}1.0\;{\mu}M$ from 100 ${\mu}M$ SNAP at 4 hours in phenol red free Hanks' balanced salt solution. But nitrite was not detectable from SNP upto 1 mM The presence of SNAP did not affect the time dependent generation of hydrogen peroxide by GO in phenol red free Hanks' balanced salt solution. Conclusion : Hydrogen peroxide generated by GO causes oxidant injury in RLMVC. Exogenous NO from NO donor prevents oxidant injury, and the protective effect may be related to the ability to release NO. These results suggest that the exogenous NO may be protective on oxidant injury to the endothelium.

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Sex Differentiation of the Gonad in Red Sea Bream, Pagrus major with Cultured Condition (양식산, 참돔 Pagrus major의 생식소 성분화)

  • 김형배
    • Journal of Aquaculture
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    • v.11 no.4
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    • pp.529-546
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    • 1998
  • Gonadal part that developed by indifferentiation period for 6 months after hatching is made as gonad and fat body. These gonad are thin semi-transparant and undistinguished germ cell. Germinal epithelium is distinguished by development of gonad epithelial tissue from 7 months after hatching. Sex differentiation is begun by oogonia develoment at 8 months after hatching. Primary oocytes grow over germinal epithelium of gonadal cavity, at 9 months after hatching, gonadal cavity become ovarian cavity as they increasing. As soon as oocytes at 13 months after hatching are filled with the whole part of gonad, degeneration of oocyte is begun. And then, gonad has cavity tissue, a small number of oocyte are located in gonadal cavity. At 15 months after hatching, new primary oocyte develop and cavity of ovarian tissue in the central of ovarian cavity. Spermatogonia multiplicate and cavity tissue consist of testicular tissue. These gonad become hermaphrodite and then ditermine the sex of female and male. These results show the red sea bream is juvenile hermaphrodite and undif-ferentiated gonochoristic teleost. Male and female differentiation type of gonad is divided in undifferentiation stage, oogonia-like stage, ovary-like stage, ovary development stage, hermaphroditic testis stage, hermaphroditic ovary stage, and testis development stage. Undifferentiation stage is continued total lenth 18cm at 13 months after hatching. ovary-like stage is continued total length 11~18cm at 13 months after hatching. Ovary-like stage is continued total length 14~26cm at 10~14 months after hatching. Ovary development stage begins from total length 20cm, 14 months after hatching. At 20 months after hatching, 44 percent of total sampled individuals had ovary. Hermaphroditic ovary stage first begins total length 19~20 cm at 15 months after hatching, but it is not observed total length 28~29cm at 20months after hatching. Hermaphroditic testis stage first begins total length 21~22cm at 20months after hatching and is continued for 20months. Testis development stage first begins total length 20~21cm at 20 months after hatching, and is occupied 33 percent total length 28~29cm at 20 months. The beginning of sex differentiation more than 50 percent is from total length 16cm at 11 months after hatching. Sex determination begins total length 20cm, 14months after hatching in female and total length 20cm, 15 months after hatching in male. Sex determination more than 50 percent begins total length 23cm,, 17 months after hatching. Undifferentiated gonadal part of red sea bream consist gonad and fat body. As differentiation is going on and gonad is growing, fat body shrinks. This appearence is showed the same tendency in 3-year old red sea bream. 1.9mm larvae after hatching grow about 19mm larvae for 47 days. The relationship between the total length and body weight of larvae and juveniles in $BW=4.45{\times}10^{-6}TL^{3.4718}$ r=0.9820. Fishes in cage culture grow to maximum total length 28.4cm. The relationship between the total length and body weight of these fishes is $BW=2.36{\times}10^{-2}TL^{2.9180}$, r=0.9971. Undifferentiated gonadal part of red sea bream consist gonad and fat body. As differentiation is going on and gonad is growing, fat body shrinks.

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