• Macromolecular Research
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• v.17 no.6
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• pp.424-429
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• 2009

In this study, we demonstrate that the biological unnatural amino acid incorporation method can be utilized in vivo to synthesize an alkyne-terminated telechelic protein, Synthesis of terminally-functionalized polymers such as telechelic polymers is recognized to be important, since they can be employed usefully in many areas of biology and material science, such as drug delivery, colloidal dispersion, surface modification, and formation of polymer network. The introduction of alkyne groups into polymeric material is particularly interesting since the alkyne group can be a linker to combine other materials using click chemistry. To synthesize the telechelic recombinant protein, we attempted to incorporate the L-homopropargylglycine into the recombinant GroES fragment by expressing the recombinant gene encoding Met at the codons for both N- and C-terminals of the protein in the Met auxotrophic E. coli via Hpg supplementation. The Hpg incorporation rate was investigated and the incorporation was confirmed by MALDI-TOF analysis of the telcchelic recombinant protein.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.390-396
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• 2013

Biosurfactants have versatile properties and potential industrial applications. A new producer, B. subtilis TU2, was isolated from the underground oil-extraction wastewater of Shengli Oilfield, China. Preliminary flask culture showed that the titer of biosurfactant obtained from the broth of TU2 was ~1.5 g/l at 48 h (718 mg/l after purification), with a reduced surface tension of 32.5 mN/m. The critical micelle concentration was measured as 50 mg/l and the surface tension maintained stability in solution with 50 g/l NaCl and 16 g/l $CaCl_2$ after 5 days of incubation at $70^{\circ}C$. FT-IR spectra exhibited the structure information of both glycolipid and lipopeptide. MALDI-TOF-MS analyses confirmed that the biosurfactant produced by B. subtilis TU2 was a blend of glycolipid and lipopeptide, including rhamnolipid, surfactin, and fengycin. The blended biosurfactant showed 86% of oil-washing efficiency and fine emulsification activity on crude oil, suggesting its potential application in enhanced oil recovery.

• Proceedings of the Korean Institute of IIIuminating and Electrical Installation Engineers Conference
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• 2004.05a
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• pp.281-284
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• 2004

Mean energy of electrons in $SF_6-Ar$ Mixtures Gas used by MCS-BEq algorithm has been analysed over the E/N range $30{\sim}300[Td]$ by a two term Boltzmann equation and by a Monte Carlo Simulation using a set of electron cross sections determined by other authors, experimentally the electron swarm parameters for 0.2[%] and 0.5[%] $SF_6-Ar$, 0.1[%] and 5.0[%], $SF_6-Ar$ mixtures were measured by time-of-flight(TOF) method. The transport Coefficients for electrons in (100[%])$SF_6$. (100[%])Ar, (0.2[%])$SF_6-Ar$ and (0.5[%]) $SF_6-Ar$, (5.0[%]) $SF_6-Ar$, (0.1[%])$SF_6-Ar$ mixtures were measured by time-of-flight method, and the electron energy distribution function and the parameters of the velocity and the diffusion were determined by the variation of the collision cross-sections with energy. The results obtained from Boltzmann equation method and Monte Carlo simulation have been compared with present and previously obtained data and respective set of electron collision cross sections of the molecules.

• Journal of Chest Surgery
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• v.32 no.5
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• pp.471-479
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• 1999

Background: Patients with tetralogy of Fallot(TOF), pulmonary atresia(PA) and major aortopulmonary collateral arteries(MAPCAs) have been managed by heterogenous surgical strategies. We have taken 5 different surgical approaches to this lesion. In this study, relative advantages and disadvantages of each strategy were discussed by analyzing our surgical results. Material and Method: Between January 1986 and June 1998, 50 patients aged 1 to 177(39${\pm}$31) months at a various morphologic spectrum of this lesion were treated at our institution. The groups, which were classified according to the initial approaches, consisted of one-stage unifocalization(A-1, n=9), staged unifocalization(A-2, n=11), right ventricle to pulmonary artery connection(RV-PA)(B-1, n=11), one-stage RV-PA plus unifocalization (B-2, n=11), and one-stage definitive repair(C, n=8). Morphologic charateristics, operative mortalities, and probabilities of definitive repair were compared between the groups and the causes of death, complications and the follow-up results were described. Result: Mean ages at the first operation were 57${\pm}$18(A-1), 42${\pm}$48(A-2), 18${\pm}$14(B-1), 52${\pm}$55(B-2), and 32${\pm}$34(C) months (p<0.05). Mean numbers of MAPCAs were least in group C (A-1=4.3${\pm}$1.0, A-2=4.5${\pm}$1.3, B-1=4.1${\pm}$1.9, B-2=4.1${\pm}$1.6, C=3.4${\pm}$1.8 : p<0.05). The ratios of the direct and the indirect MAPCAs in each group were not different between the grousps (A-1=91%/9%, A-2=78%/22%, B-1=80%/20%, B-2=80%/20%, C=81%/19% : p>0.05). Nineteen patients had more than 1 dependent MAPCAs. True pulmonary arteries were not present 13 patients and they were confluent in 29. A total of 101 operations were performed. Operative mortalities of initial procedures were 33%(3/9, A-1), 18%(2/11, A-2), 0%(0/11, B-1), 36%(5/11, B-2) and 13%(1/8, C) (p<0.05). Percentages of the definitive repair among the patients of each group were 22%(2/9, A-1), 18%(2/11, A-2). 45%(5/11, B-1), 27%(4/11, B-2), and 100%(8/8, C)(p<0.05). Hypoxic respiratory and cardiac failure(6), hypoxic encephalopathy(2) and sepsis(4) were the major causes of death. Phrenic nerve palsy occured in 5 patients. Thirty-one patients among the survivals have been followed up for a mean duration of 74${\pm}$42(3-145) months. One and five year actuarial survival rates were 73% and 73 %. Conclusion: In conclusion, Right ventricle to pulmonary artery connection used in patients with TOF/PA/MAPCA as an inital procedure appeared to be highly successful in enhancing the chance of satisfactory definitive repair without the significant surgical risks. One-stage total repair at an ealier age group could be performed safely with the resonable outcomes. Unifocalization approach, whether it was performed in a single stage or in the multiple stages, resulted in the high operative mortality and the lowest chance of definitive repair, however more tailored selection of the patients and the long follow-up is mandatory to prove the usefulness of this approach.

• Analytical Science and Technology
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• v.28 no.2
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• pp.132-138
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• 2015

Rosiglitazone metabolites in rat plasma were analyzed after intraperitoneal and oral administration to rats. Seven metabolites (M1-M7) were detected in rat plasma (IP and PO), and the structures were confirmed using liquid chromatography-triple time of flight (TOF) mass spectrometry; as a result, the most abundant metabolite was M5, a de-methylated rosiglitazone. Other minor in vivo metabolites were driven from monooxygenation and demethylation (M2), thiazolidinedione ring-opening (M1, M3), mono-oxygenation (M4, M7), and mono-oxygenation followed by sulfation (M6). Among them, M1 was found to be a 3-{p-[2-(N-methyl-N-2-pyridylamino)ethoxy]phenyl}-2-(methylsulfinyl)propionamide, which is a novel metabolite of rosiglitazone. There was no significant difference in the metabolic profiles resulting from the two administrations. The findings of this study provide the first comparison of circulating metabolite profiles of rosiglitazone in rat after IP and PO administration and a novel metabolite of rosiglitazone in rat plasma.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1076-1080
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• 2008

Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat ${\alpha}$-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.219-227
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• 2008

The free-living photoheterotrophic Gram-negative bacterium Rhodobacter sphaeroides possesses a quorum-sensing (QS) regulatory system mediated by CerR-CerI, a member of the LuxR-LuxI family. To identify the genes affected by the regulatory system, random lacZ fusions were generated in the genome of R. sphaeroides strain 2.4.1 using a promoter-trapping vector, pSG2. About 20,000 clones were screened and 23 showed a significantly different level of ${\beta}$-gal activities upon the addition of synthetic 7,8-cis-N-tetradecenoyl-homoserine lactone (RAI). Among these 23 clones, the clone showing the highest level of induction was selected for further study, where about a ten-fold increase of ${\beta}$-gal activity was exhibited in the presence of RAI and induction was shown to be required for cerR. In this clone, the lacZ reporter was inserted in a putative gene that exhibited a low homology with catD. A genetic analysis showed that the expression of the catD homolog was initiated from a promoter of another gene present upstream of the catD. This upstream gene showed a strong homology with luxR and hence was named qsrR (quorum-sensing regulation regulator). A comparison of the total protein expression profiles for the wild-type cells and qsrR-null mutant cells using two-dimensional gel electrophoresis and a MALDI-TOF analysis allowed the identification of sets of genes modulated by the luxR homolog.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1306-1318
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• 2009

The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes. Two $\alpha$-amylases ScAmy54 and ScAmy43 predicted to play an important role in starch degradation were showed to produce specific oligosaccharides essentially maltotriose that have a considerable commercial interest. Primary structure of the two enzymes was established by N-terminal sequencing, MALDI-TOF masse spectrometry and cDNA cloning. The two proteins have the same N-terminal catalytic domain and ScAmy43 derived from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. Data of genomic analysis suggested that the two enzymes originated from the same $\alpha$-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during S. sclerotiorum cultivation. The structural gene of Scamy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 residues. ScAmy54 exhibited high amino acid homology with other liquefying fungal $\alpha$-amylases essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3-D structure of 2guy from A. niger as template. ScAmy54 is composed by three domains A, B, and C, including the well-known $(\beta/\alpha)_8$ barrel motif in domain A, have a typical structure of $\alpha$-amylase family, whereas ScAmy43 contained only tow domains A and B is the first fungal $\alpha$-amylase described until now with the smallest catalytic domain.

• Proceedings of the Korean Vacuum Society Conference
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• 2012.02a
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• pp.257-258
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• 2012

The SERS spectra of 4-aminobenzenethiol (ABT) have served as the "probe" molecule, which have helped spectroscopists to build up the electromagnetic (EM) and chemical (CHEM) enhancement mechanisms. In particular, the b2-peaks (9b, 3, and 19b) of the SERS spectra of ABT have been attributed to arise from the vibronic charge-transfer (CT) between Au or Ag surface and the ABT. Quite recently, however, Tian and co-workers [1] claimed that the b2-peaks are not the CT-enhanced spectra of ABT. Instead, these peaks arise from the 4,4'-dimercaptoazobenzenes (DMABs) that are produced by the oxidative coupling of two ABTs. Their claim is under intense debate currently. Herein, we studied spatially and temporally resolved SERS spectra of ABTs on Ag thin film (thickness of 10 nm), to investigate such claim. Herein, we present a series of additional evidences that strongly support that the b2 intensities of ABTs do not arise from the CT-enhancement: (1) the b2-peaks can be locally "activated" (i. e. turned on) irreversibly with focused laser radiation; (2) the TOF-SIM spectrometry on the activated region show depletion of ABT-Ag+ ions; and finally (3) the spatially resolved FT-IR spectra of the activated region show two pronounced peaks at 1377 cm-1 and 1460 cm-1, both of which can be assigned to the stretching mode of N=N bond. While the result does not disprove the existence of CT or CHEM enhancement in general, the results do show that previous interpretations of the spectra of ABTs should be re-interpreted.