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PHUE, JE-NIE,OH, SUNG-JIN,SON, YOUNG-JIN,KIM, YONG-IN,KIM, KYUNG-HWAN,KIM, JUNG-WOO,HONG, CHUNG IL,CHUNG, IN-SIK,HAHN, TAE-RYUNG 경희대학교 생명자원과학연구원 2000 硏究論文集 Vol.21 No.-
An improved method of refolding recombinant human promsulin from E. coli was presented. It was based on a two-stage stirred tank reactor in which denatured promsulins-sulfonate was mixed instantaneously with a reaction buffer in the first stage reactor, and then fed to the second stage reactor. The mixture was stirred further for a total of 30h in the second stage reactor. In this system, unfavorable effects present due to the increase in reaction volume and protein concentration for protein refolding, which becomes significant in a large-scale operation, were avoided. Refolding yields of over 80% was obtained for achieving reaction volume of upto 501 at a protein concentration of 1 mg/ml. The optimum urea concentration was 1 M. Refolding yield at the 1-1 reaction volume and protein concentration of 0.5 mg/ml was increased about 2.5-fold, compared to that in a batch reactor. By increasing protein concentration in a two-stage refolding reaction, the cost for insulin production could be reduced, therefore making this process economical.
Effects of Cisplatin-DNA Adducts on RecA Mediated Strand Exchange Reaction
Phue, Je-Nie,Bae, Jun-Seong,Hahn, Tae-Ryong 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.7
The effects of cisplatin-DNA adducts on RecA mediated strand exchange reactions were examined in vitro. E. coli RecA protein catalizes strand exchange reaction between linear duplex and homologous single stranded DNA molecules to produce nicked circular heteroduplex DNA which can be detected by agarose gel electrophoresis. Platinum binding to both of DNA substrates inhibits significantly strand exchange reaction mediated by RecA protein. The exchanged heteroduplex DNA products were drastically decreased when the linear duplex DNA was treated with cisplatin. Those were not detected when the single stranded DNA was treated with cisplatin under the same conditions. These results suggest that single stranded DNA is more sensitive to cisplatin than duplex and/or that platinum-DNA adducts in single strands are structurally different from those of duplex.
Phue, Je-Nie,Hahn, Tae-Ryong 경희대학교 유전공학연구소 1990 遺傳工學論文集 Vol.2 No.-
대장균의 RecA 단백질은 circular single stranded DNA와 linear duplex DNA의 homologous pairing 및 이들 사이의 strand 교환을 촉진시킨다. 이 strand 교환반응의 결과, nicked heteroduplex DNA와 교환반응에 참여하지 않은 linear single stranded DNA가 생성된다. 따라서, DNA agarose 겔 전기영동상에서 형성된 nicked heteroduplex DNA의 band로 strand 교환반응을 관찰할 수 있다. RecA 단백질의 순수분리에는 recA 유전자를 가지고 있는 plasmid가 들어있는 E coli 균주 KM4104를 사용하였다. RecA 단백질의 분리는 Polyamin P precipitation, ammonium sulfate fractionation, hydroxylapatite chromatography 및 ssDNA agarose chromatography를 이용하였다. DNA 기질상의 insertion과 deletion이 circular single stranded DNA상에 존재하고 있을 때, RecA 단백질은 120 unpaired bases까지 안정한 heteroduplex DNA를 형성할 수 있었다. 그러나 insertion이 linear duplex DNA상에 존재했을 경우 38 unpaired bases에서는 교환산물이 생성되었지만 120 unpaired bases에서는 관찰되지 않았다. RecA protein from E. coli promotes the homologous paring and strand exchange between single stranded circular and homologous linear duplex DNAs. This strand exchange reaction generates nicked circular heteroduplex and linear single stranded DNAs. Thus, the completion of strand exchange can be demonstrated by the production of nicked circular heteroduplex DNA which can be detected by gel electrophoresis. E. coli strain KM4104 which contains multicopy plasmids carrying E. coli recA gene was used for the purification of RecA protein. Purification steps include polyamin P precipitation, ammonium sulfate fractionation, hydroxylapatite chromatography and ssDNA agarose chromatography. Effects of deletions and insertions in DNA substrates on RecA mediated strand exchange reactions were investigated. RecA catalyzed strand exchange to form stable heteroduplex products with upto 120 unpaired bases when the insert is located within a single stranded circular molecule. However, when the insert is located in the linear duplex DNA substrates, exchange products were formed with 38 unpaired bases but not detected with 120 base pairs of insertions.
Phue, Je Nie,Oh, Sung Jin,Son, Young Jin,Kim, Yong In,Kim, Kyung Hwan,Kim, Jung Woo,Hong, Chung Il,Chung, In Sik,Hahn, Tae Ryong 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.1
An improved method of refolding recombinant human proinsulin from E. coli was presented. It was based on a two-stage stirred tank reactor in which denatured proinsulins-sulfonate was mixed instantaneously with a reaction buffer in the first stage reactor, and then fed to the second stage reactor. The mixture was stirred further for a total of 30h in the second stage reactor. In this system, unfavorable effects present due to the increase in reaction volume and protein concentration for protein refolding, which becomes significant in a large-scale operation, were avoided. Refolding yields of over 80% was obtained for achieving reaction volume of upto 50 l at a protein concentration of 1㎎/㎖. The optimum urea concentration was 1M. Refolding yield at the 1-1 reaction volume and protein concentration of 0.5㎎/㎖ was increased about 2.5-fold, compared to that in a batch reactor. By increasing protein concentration in a two-stage refolding reaction, the cost for insulin production could be reduced, therefore, making this process economical.
Effects of Cisplatin - DNA Adducts on RecA Mediated Strand Exchange Reaction
Je Nie Phue,Jun Seong Bae,Tae Ryong Hahn 생화학분자생물학회 1992 BMB Reports Vol.25 No.7
The effects of cisplatin-DNA adducts on RecA mediated strand exchange reactions were examined in vitro. E. coli RecA protein catalizes strand exchange reaction between linear duplex and homologous single stranded DNA molecules to produce nicked circular heteroduplex DNA which can be detected by agarose gel electrophoresis. Platinum binding to both of DNA substrates inhibits significantly strand exchange reaction mediated by RecA protein. The exchanged heteroduplex DNA products were drastically decreased when the linear duplex DNA was treated with cisplatin. Those were not detected when the single stranded DNA was treated with cisplatin under the same conditions. These results suggest that single stranded DNA is more sensitive to cisplatin than duplex and/or that platinum-DNA adducts in single strands are structurally different from those of duplex.
부제니,김지영 경희대학교 유전공학연구소 1993 遺傳工學論文集 Vol.5 No.-
Oxidative stress is one of the most important causative agents of mutagenesis, aging. carcinogenesis and a number of diseases(Farr & Kogoma, 1991). Active oxygen species oxidize fatty acid, protein and carbohydrates, and finally attack DNA and deoxynucleotides. Virtually all aerobic organisms have developed complex defense and repair mechanisms to mitigate the deleterious effects of active oxygen species.
Two-Photon Dye Cocktail for Dual-Color 3D Imaging of Pancreatic Beta and Alpha Cells in Live Islets
Agrawalla, Bikram Keshari,Lee, Hyo Won,Phue, Wut-Hmone,Raju, Anandhkumar,Kim, Jong-Jin,Kim, Hwan Myung,Kang, Nam-Young,Chang, Young-Tae American Chemical Society 2017 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.139 No.9
<P>Insulin-secreting beta cells together with glucagon-producing alpha cells play an essential role in maintaining the optimal blood glucose level in the body, so the development of selective probes for imaging of these cell types in live islets is highly desired. Herein we report the development of a 2glucosamine-based two-photon fluorescent probe, TP-beta, that is suitable for imaging of beta cells in live pancreatic islets from mice. Flow cytometry studies confirmed that TP-beta is suitable for isolation of primary beta cells. Moreover, two-photon imaging of TP-beta-stained pancreatic islets showed brightly stained beta cells in live islets. Insulin enzyme-linked immunosorbent assays revealed that 'TP-beta has no effect on glucose-stimulated insulin secretion from the stained islet. Finally, to develop a more convenient islet imaging application, we combined our recently published alpha-cell-selective probe TP-alpha with TP-beta to make a 'TP islet cocktail'. This unique dye cocktail enabled single excitation (820 nm) and simultaneous dual-color imaging of alpha cells (green) and beta cells (red) in live pancreatic islets. This robust TP islet cocktail may serve as a valuable tool for basic diabetic studies.</P>
Johnson Jesse A.,Kashka F. Mallari,Pepe Vincent M.,Treacy Taylor,McDonough Gregory,Khaing Phue,McGrath Christopher,George Brandon J.,Yoo Erika J. 대한중환자의학회 2023 Acute and Critical Care Vol.38 No.3
Background: There is increasing heterogeneity in the clinical phenotype of patients admitted to the intensive care unit (ICU) with coronavirus disease 2019 (COVID-19), and reasons for mechanical ventilation are not limited to COVID pneumonia. We aimed to compare the characteristics and outcomes of intubated patients admitted to the ICU with the primary diagnosis of acute hypoxemic respiratory failure (AHRF) attributed to COVID pneumonia with those of patients admitted for an alternative diagnosis.Methods: This was a retrospective cohort study of adults with confirmed severe acute respiratory syndrome coronavirus 2 infection admitted to one of nine ICUs between March 18, 2020, and April 30, 2021, at an urban university institution. We compared characteristics between the two groups using appropriate statistics and performed logistic regression to identify risk factors for death in the mechanically ventilated COVID-19 population. Results: After exclusions, the final sample consisted of 319 patients with respiratory failure secondary to COVID pneumonia and 150 patients intubated for alternative diagnoses. The former group had higher rates of ICU mortality (57.7% vs. 36.7%, P<0.0001) and hospital mortality (58.9% vs. 39.3%, P<0.0001). Patients with AHRF secondary to COVID-19 pneumonia also had longer lengths of stay in the ICU (12 vs. 6 days, P<0.0001) and hospital (20 vs. 13.5 days, P=0.0001). Following risk adjustment, these COVID-19 patients had 2.25 times greater odds of death (95% confidence interval, 1.42¬–3.56; P=0.001).Conclusions: Mechanically ventilated patients admitted to the ICU with COVID-19–associated respiratory failure are at higher risk of hospital death and have worse utilization outcomes than those whose reason for ICU admission is unrelated to COVID pneumonia.