• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.537-543
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• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.620-627
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• 2004

On the foundation of a database of genome sequences and protein analyses, the ability to clone a gene based on a peptide analysis is becoming more feasible and effective for identifying a specific gene and its protein product of interest. As such, the current study conducted a protein analysis using 2-D PAGE followed by MALDI- TOF and ESI-MS to identify a highly expressed gene product of C. parasitica. A distinctive and highly expressed protein spot with a molecular size of 47.2 kDa was randomly selected and MALDI-TOF MS analysis was conducted. A homology search indicated that the protein appeared to be a fungal enolase (enol). Meanwhile, multiple alignments of fungal enolases revealed a conserved amino acid sequence, from which degenerated primers were designed. A screening of the genomic $\lambda$ library of C. parasitica, using the PCR amplicon as a probe, was conducted to obtain the full-length gene, while RT-PCR was performed for the cDNA. The E. coli-expressed eno 1 exhibited enolase enzymatic activity, indicating that the cloned gene encoded the C. parasitica enolase. Moreover, ESI-MS of two of the separated peptides resolved from the protein spot on 2-D PAGE revealed sequences identical to the deduced sequences, suggesting that the cloned gene indeed encoded the resolved protein spot. Northern blot analysis indicated a consistent accumulation of an eno1 transcript during the cultivation.

• Molecules and Cells
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• v.41 no.10
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• pp.900-908
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• 2018

Insulin resistance is closely associated with metabolic diseases such as type 2 diabetes, dyslipidemia, hypertension and atherosclerosis. Thiazolidinediones (TZDs) have been developed to ameliorate insulin resistance by activation of peroxisome proliferator-activated receptor (PPAR) ${\gamma}$. Although TZDs are synthetic ligands for $PPAR{\gamma}$, metabolic outcomes of each TZD are different. Moreover, there are lack of head-to-head comparative studies among TZDs in the aspect of metabolic outcomes. In this study, we analyzed the effects of three TZDs, including lobeglitazone (Lobe), rosiglitazone (Rosi), and pioglitazone (Pio) on metabolic and thermogenic regulation. In adipocytes, Lobe more potently stimulated adipogenesis and insulin-dependent glucose uptake than Rosi and Pio. In the presence of pro-inflammatory stimuli, Lobe efficiently suppressed expressions of pro-inflammatory genes in macrophages and adipocytes. In obese and diabetic db/db mice, Lobe effectively promoted insulin-stimulated glucose uptake and suppressed pro-inflammatory responses in epididymal white adipose tissue (EAT), leading to improve glucose intolerance. Compared to other two TZDs, Lobe enhanced beige adipocyte formation and thermogenic gene expression in inguinal white adipose tissue (IAT) of lean mice, which would be attributable to cold-induced thermogenesis. Collectively, these comparison data suggest that Lobe could relieve insulin resistance and enhance thermogenesis at low-concentration conditions where Rosi and Pio are less effective.

• Animal cells and systems
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• v.1 no.3
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• pp.521-526
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• 1997

We isolated a mouse nck cDNA from the thymus cDNA expression library. The cDNA encodes a 377 amino acid protein and displays 97% amino acid sequence identity to human oncogenic protein nck, which is composed almost exclusivelv of three src homology 3 (SH3) domains and one SH2 domain. The sequence analysis also showed that the isolated cDNA is the mouse counterpart of the human nck and different from the mouse grb4, which has been reported to be highly similar to the human nck and, therefore considered as a mouse nck, Northern blot analysis showed that the transcript of the gene was 1.8 kb and was highly expressed in the testis, thymus, and brain but moderately in the liver and lymph node. Western blot analysis showed that the size of the protein was about 47 kDa. Overexpression of the mouse Nck transformed a mouse fibroblast cell line, NIH3T3. The results clearly indicate that normal nck gene has transforming ability and provide an argument against a suggested possibility that the transforming ability of the human nck gene is due to a mutation(s) in the gene.

• BMB Reports
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• v.37 no.3
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• pp.292-297
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• 2004

The current model for the mechanism of action of the Bacillus thuringiensis Cry $\delta$-endotoxins involves the penetration of the ${\alpha}4-{\alpha}5$ hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the ${\alpha}4-{\alpha}5$ loop of the 130-kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the ${\alpha}4-{\alpha}5$ loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the ${\alpha}4-{\alpha}5$ loop of the Cry4A toxin.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.531-531
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• 2005

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.509-509
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• 2005

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.508-508
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• 2005

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2003.06a
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• pp.196-197
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• 2003

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.485-485
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• 2005