• Title/Summary/Keyword: ${\beta}$-Hydrogen elimination

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Effects of $\beta$-Mercaptoethanol and Hydrogen Peroxide on Enzymatic Conversion of Human Proinsulin to Insulin

  • Son, Young-Jin;Kim, Chang-Kyu;Choi, Byoung-Taek;Park, Yong-Cheol;Seo, Jin-Ho
    • Journal of Microbiology and Biotechnology
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    • v.18 no.5
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    • pp.983-989
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    • 2008
  • Human insulin is a hormone well-known to regulate the blood glucose level. Recombinant preproinsulin, a precursor of authentic insulin, is typically produced in E. coli as an inactive inclusion body, the solubilization of which needs the addition of reducing agents such as $\beta$-mercaptoethanol. To make authentic insulin, recombinant preproinsulin is modified enzymatically by trypsin and carboxypeptidase B. The effects of $\beta$-mercaptoethanol on the formation of human insulin derivatives were investigated in the enzymatic modification by using commercially available human proinsulin as a substrate. Addition of 1 mM $\beta$-mercaptoethanol induced the formation of various insulin derivatives. Among them, the second major one, impurity 3, was found to be identical to the insulin B chain fragment from $Phe_1$ to $Glu_{21}$. Minimization of the formation of insulin derivatives and concomitant improvement of the production yield of human insulin were achieved by the addition of hydrogen peroxide. Hydrogen peroxide bound with $\beta$-mercaptoethanol and thereby reduced the negative effects of $\beta$-mercaptoethanol considerably. Elimination of the impurity 3 and other derivatives by the addition of over 10 mM hydrogen peroxide in the presence of $\beta$-mercaptoethanolled to a 1.3-fold increase in the recovery efficiency of insulin, compared with those for the case without hydrogen peroxide. The positive effects of hydrogen peroxide were also confirmed with recombinant human preproinsulin expressed in recombinant E. coli as an inclusion body.

Non-stoichiometric AlOx Films Prepared by Chemical Vapor Deposition Using Dimethylaluminum Isopropoxide as Single Precursor and Their Non-volatile Memory Characteristics

  • Lee, Sun-Sook;Lee, Eun-Seok;Kim, Seok-Hwan;Lee, Byung-Kook;Jeong, Seok-Jong;Hwang, Jin-Ha;Kim, Chang-Gyoun;Chung, Taek-Mo;An, Ki-Seok
    • Bulletin of the Korean Chemical Society
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    • v.33 no.7
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    • pp.2207-2212
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    • 2012
  • Dimethylaluminum isopropoxide (DMAI, $(CH_3)_2AlO^iPr$) as a single precursor, which contains one aluminum and one oxygen atom, has been adopted to deposit non-stoichiometric aluminum oxide ($AlO_x$) films by low pressure metal organic chemical vapor deposition without an additional oxygen source. The atomic concentration of Al and O in the deposited $AlO_x$ film was measured to be Al:O = ~1:1.1 and any serious interfacial oxide layer between the film and Si substrate was not observed. Gaseous by-products monitored by quadruple mass spectrometry show that ${\beta}$-hydrogen elimination mechanism is mainly contributed to the $AlO_x$ CVD process of DMAI precursor. The current-voltage characteristics of the $AlO_x$ film in Au/$AlO_x$/Ir metalinsulator-metal (MIM) capacitor structure show high ON/OFF ratio larger than ${\sim}10^6$ with SET and RESET voltages of 2.7 and 0.8 V, respectively. Impedance spectra indicate that the switching and memory phenomena are based on the bulk-based origins, presumably the formation and rupture of filaments.

Nucleophilic Displacement at Sulfur Center (XI). Reaction of Naphthalenesulfonyl Chlorides with Anilines (황의 친핵성 치환반응 (제11보). 염화나프탈렌술포닐과 아닐린과의 반응)

  • Lee Ik Chon;Uhm Tae Seop;Lee Euk Suk
    • Journal of the Korean Chemical Society
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    • v.22 no.5
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    • pp.281-288
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    • 1978
  • Pseudo-first order rate constants $k_{obs}$ were determined for the reactions of naphthalenesulfonyl chlorides (1-NSC and 2-NSC) and anilines. The second order rate constant $k_2$ and third order catalytic $k_3$ were then determined from $k_{obs}$. For 1-NSC peri-hydrogen effect was observed. The large Brønsted ${\beta}$ and large negative slopes ${\rho}$ for the Hammett plots were obtained. These results with the unsually low values of activation parameters were consistent with the $S_AN$-elimination mechanism, but these can be equally well interpreted with the associative $S_N2$mechanism.

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A Molecular Modeling Study of AAD16034

  • Cho, Hoon;Choi, Cheol-Hee;Yoo, Kyung-Ho;Cho, Seung-Joo
    • Molecular & Cellular Toxicology
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    • v.4 no.4
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    • pp.307-310
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    • 2008
  • AAD16034 is an alginate lyase from Pseudoalteromonas sp. IAM14594. A very close homologue with known 3D structure exists (marine bacterium Pseudoalteromonas sp. strain no. 272). A three-dimensional structure of AAD16034 was generated based on this template (PDB code: 1J1T) by comparative modeling. The modeled enzyme exhibited a jelly-roll like structure very similar to its template structure. Both enzymes possess the characteristic alginate sequence YFKhG+Y-Q. Since AAD16034 displays enzymatic activity for poly-M alginate, docking of a tri-mannuronate into the modeled structure was performed. Two separate and adjacent binding sites were found. The ligand was accommodated inside each binding site. By considering both binding sites, a plausible binding pose for the poly-M alginate polymer could be deduced. From the modeled docking pose (i.e., the most important factor that attracts alginate polymer into this lyase) the most likely interaction was electrostatic. In accordance with a previous report, the hydroxyl group of Y345 was positioned close to the ${\alpha}$-hydrogen of ${\beta}$-mannuronate, which was suitable to initiate a ${\beta}$-elimination reaction. K347 was also very near to the carboxylatemoiety of the ligand, which might stabilize the dianion intermediate during the ${\beta}$-elimination reaction. This implies that the characteristic alginate sequence is absolutely crucial for the catalysis. These results may be exploited in the design of novel enzymes with desired properties.

Antioxidative and Anti-proliferative Effects on MCF-7 Human Breast Cancer Cells of Ikiyangyoung-Tang (익기양영탕(益氣養榮湯)의 항산화 및 유방암 세포주 생장 억제 효과에 미치는 영향)

  • Park, Kyung-Mi;Cho, Seong-Hee;Lee, Jin-A
    • The Journal of Korean Obstetrics and Gynecology
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    • v.20 no.1
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    • pp.32-49
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    • 2007
  • Purpose: Breast cancer is the most common disease in Korean women. Despite remarkable improvements in treatment strategies against various cancer during the past 40 years, breast cancer still remains as one of the main causes of cancer mortality among women in the whole world. This study was carried out to investigate antioxidative and anti-proliferative effects on MCF-7 human breast cancer cells of Ikiyangyoung-Tang extract. Methods: We measured a content of polyphenol and flavonoid in the Ikiyangyoung-Tang extract, eliminative ability of DPPH radical, ABTS free radical and hydrogen peroxide, antioxidative effects of linoleic acid, cytotoxicity on MCF-7 human breast cancer cells. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium/F12(DMEM/F12) supplemented with 10 % fetal bovine serum(FBS; Gibco) and antibiotics. Results : The extract of Ikiyangyoung-Tang contains polyphenol of 168.3${\pm}$12.8 ${\mu}$g/mg and flavonoid of 84.3${\pm}$3.4 ${\mu}$g/mg. Above results show profitable abilities of elimination of ${\alpha}$-${\alpha}$-Diphenyl-${\beta}$-picrylhydrazyl(DPPH) radical, ABTS free radical and hydrogen peroxide. Also, the extract of Ikiyangyoung-Tang strongly inhibits the proliferation of MCF-7 cells in a dose ependent manner. And. it has cytotoxicity on NIH3T3 cells. Conclusion : It can be concluded that Ikiyangyoung-Tang extract has an antioxidative effect and antiproliferative effect on MCF-7 human breast cancer cells.

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