• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.136-139
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• 2009

Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. Previously, comparative transcriptome analysis between the low-producer S. avermitilis ATCC31267 and the high-producer S. avermitilis ATCC31780 using a S. avermitilis whole genome chip revealed that 50 genes were overexpressed at least two-fold higher in S. avermitilis ATCC31780. To verify the biological significance of some of the transcriptomics-guided targets, five putative regulatory genes were individually cloned under the strong-and-constitutive promoter of the Streptomyces expression vector pSE34, followed by the transformation into the low-producer S. avermitilis ATCC31267. Among the putative genes tested, three regulatory genes including SAV213, SAV3818, and SAV4023 exhibited stimulatory effects on avermectin production in S. avermitilis ATCC31267. Moreover, overexpression of SAV3818 also stimulated actinorhodin production in both S. coelicolor M145 and S. lividans TK21, implying that the SAV3818, a putative TetR-family transcriptional regulator, could be a global upregulator acting in antibiotic production in Streptomyces species.

• The Korean Journal of Physiology and Pharmacology
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• 제9권3호
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• pp.159-164
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• 2005

Effects of antioxidants on the established nephropathy were investigated. The experimental nephropathy was induced in rats by intravenous injection of adriamycin (2 mg/kg). Six weeks later, when proteinuria was apparent, the rats were supplemented with N-acetylcysteine (NAC, 1 g/kg/day) in drinking water for additional 6 weeks. Glomerulosclerosis score and tubulointerstitial injury index were determined by light microscopy. Expression of transforming growth factor (TGF) ${\beta}1$ and laminin ${\beta}1$ was determined in the renal cortex by reverse transcription-polymerase chain reaction, Western blotting, immunohistochemistry, and immunogold electron microscopy. The adriamycin-induced proteinuria as well as the glomerulosclerosis and tubulointerstitial injury was ameliorated by the treatment with NAC. Adriamycin increased the expression of TGF ${\beta}1$ mRNA and protein, which was ameliorated by NAC. Although the expression of laminin ${\beta}1$ mRNA was increased, adriamycin did not significantly alter that of its protein. These results indicate that antioxidants ameliorate the established nephropathy in association with normalization of overexpressed TGF ${\beta}1$.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.333-340
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• 1998

Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM -sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.738-744
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• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1699-1706
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• 2014

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-${\beta}$-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley ${\beta}$-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at $50^{\circ}C$, with Azo-barley-glucan as the substrate. The metal ions $Mn^{2+}$, $Mg^{2+}$, $Ca^{2+}$, and $Fe^{2+}$ enhanced the enzymatic activity, whereas the $Cu^{2+}$ and $Zn^{2+}$ ions inhibited the enzymatic activity. The $K_m$ and $V_{max}$ values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

• Bulletin of the Korean Chemical Society
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• 제29권10호
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• pp.2023-2025
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• 2008

To evaluate the potential of radioiodine labelled hypericin as a malignant glioma imaging agent, U-251 MG, U-373 MG, C6 glioma and fibroblast were treated with a I-123 labelled hypericin derivative and C6 glioma transplanted nude mouse were injected with a I-124 labelled hypericin derivative for a micro PET imaging. 2- Iodohypericin was prepared as a reference compound. In this paper, we describe the syntheses of 2- iodohypericin and 2-[$^{123}I/^{124}I$]iodohypericin and the results of a corresponding biological evaluation. In all glioma cell lines, 2-[$^{123}I$]iodohypericin uptake was increased in a time dependant manner and an accumulation of 2-[$^{124}I$]iodohypericin was observed in C6 glioma bearing nude mouse. These results suggest that radioiodine labelled hypericin can visualize a PKC overexpressed malignant glioma.

• IMMUNE NETWORK
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• 제10권4호
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• pp.120-125
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• 2010

Dysfunction of the virus-specific T cells is a cardinal feature in chronic persistent viral infections such as one caused by hepatitis C virus (HCV). In chronic HCV infection, virus-specific dysfunctional CD8 T cells often overexpress various inhibitory receptors. Programmed cell death 1 (PD-1) was the first among these inhibitory receptors that were identified to be overexpressed in functionally impaired T cells. The roles of other inhibitory receptors such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and T cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) have also been demonstrated in T-cell dysfunctions that occur in chronic HCV patients. Blocking these inhibitory receptors in vitro restores the functions of HCV-specific CD8 T cells and allows enhanced proliferation, cytolytic activity and cytokine production. Therefore, the blockade of the inhibitory receptors is considered as a novel strategy for the treatment of chronic HCV infection.

• International Journal of Oral Biology
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• 제39권1호
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• pp.1-7
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• 2014

Neuromedin B (NMB) acts as a growth factor or a morphogen and plays a role in cancer progression. Indeed, the NMB receptor (NMB-R) is overexpressed in different types of tumors. In our current study, we investigated the involvement of NMB-R in the proliferation of oral cancer cells. Human oral squamous cell carcinoma (SCC) and human oral cancer cells, SCC-25 cells were found to be NMB-R-positive. The NMB-R antagonist PD168368 inhibited the proliferation of SCC-25 cells and reduced their colony formation capacity. We also found that PD168368 induced the cell cycle arrest and apoptosis of SCC-25 cells in a dose-/time-dependent manner. Overall, this antitumor activity of PD168368 in human oral cancer cells suggests that NMB-R is a potential target for the future prevention and treatment of human cancers.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.243-246
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• 2005

A novel esterase showing high enantioselectivity to (S)-ketoprofen ethyl ester was selected from fosmid environmental DNA library which is provided by Microbial Genomic & Applications Center. As a result of Blast search, the gene wasn't registerated in Gene Bank yet. And as we know, conserved domain region of esterase , G-X-S-X-G, wasn't discovered.$^{4)}$ And it is similar to Beta-lactamase. The DNA sequence of cloned esterase include an open reading frame consisting of 1170 bp, designated as EST-Y29, encoding a protein of 389 amino acids with a molecular mass of about 42.8 kDa. And amino acid sequence analysis revealed only a few identity (28%) to tile known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases. when being comparison to other esterase revealed , this enzyme seems to be classified as a new member of esterase family. EST-Y29 was functionally overexpressed in a soluble form in E. coli with maximum conversion yield of (S)-ketoprofen at $65^{\circ}C$. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzyme from a metagenome.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.599-602
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• 2005

Truncated form of UBP1, an ubiquitin-specific protease of Saccharomyces cerevisiae, was overexpressed in Escherichia coli. The hexahistidine residue $(His_6)$ was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were $40^{\circ}C$ and pH 8.0, respectively.