• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1288-1294
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• 2009

We have used mutational analysis to identity four genes, MXAN3553, MXAN3554, MXAN3555, and MXAN3556, constituting an operon that is essential for normal fruiting body development in Myxococcus xanthus. Deletion of MXAN3553, which encoded a hypothetical protein, resulted in delayed fruiting body development. MXAN3554 was predicted to encode a metallopeptidase, and its deletion caused fruiting body formation to fail. Inactivation of MXAN3555, which encoded a putative NtrC-type response regulator, resulted in delayed aggregation and a severe reduction in sporulation. Fruiting bodies also failed to develop with the deletion of MXAN3556, another gene encoding a hypothetical protein.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2005.06a
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• pp.107-109
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• 2005

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.57-57
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• 1999

It is well known that rapidly activating delayed rectifier $K^{＋}$ channels ( $I_{Kr}$ ) playa role in repolarisation in mammalian hearts. Recently, human ether-a- go- go related gene (HERG) channels was shown to be a molecular equivalent to $I_{Kr}$ . We have investigated the permeation of various external cations on $I_{Kr}$ in mammalian hearts and on HERG channels expressed in Xenopus laevis oocytes.(omitted)

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.932-945
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• 2009

Several species of Gram-positive bacteria have cell wall peptidoglycan (syn. murein) in which not all of the sugar moieties are N-acetylated. This has recently been shown to be a secondary effect, caused by the action of a peptidoglycan N-acetylglucosamine deacetylase. We have found that the opportunistic pathogen Listeria monocytogenes is unusual in having three enzymes with such activity, two of which remain in the cytoplasm. Here, we examine the enzyme (PgdA) that crosses the cytoplasmic membrane and is localized in the cell wall. We purified a hexa-His-tagged form of PgdA to study its activity and constructed a mutant devoid of functional Lmo0415 (PgdA) protein. L. monocytogenes PgdA protein exhibited peptidoglycan N-acetylglucosamine deacetylase activity with natural substrates (peptidoglycan) from both L. monocytogenes and Escherichia coli as well as the peptidoglycan sugar chain component N-acetylglucosamine, but not with N-acetylmuramic acid. As was reported recently [6], inactivation of the structural gene was not lethal for L. monocytogenes nor did it affect growth rate or morphology of the cells. However, the pgdA mutant was more prone to autolysis induced by such agents as Triton X-100 and EDTA, and is more susceptible to the cationic antimicrobial peptides (CAMP) lysozyme and mutanolysin, using either peptidoglycan muramidases or autolysis-inducing agents. The pgdA mutant was also slightly more susceptible than the wild-type strain to the action of certain beta-lactam antibiotics. Our results indicate that protein PgdA plays a protective physiological role for listerial cells.

• Biomolecules & Therapeutics
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• v.16 no.4
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• pp.385-393
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• 2008

As an attempt to search for bioactive natural products exerting anti-inflammatory activity, we evaluated the effects of the methanol extract of Polytrichum commune Hedw (PCM) (Polytrichaceae) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin $E_2$ ($PGE_2$) and pro-inflammatory cytokines release in murine macrophage cell line RAW 264.7. PCM potently inhibits the production of NO, $PGE_2$, tumor necrosis factor (TNF)-$\alpha$ and interleukin (IL)-6. Consistent with these results, PCM also concentration-dependently inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygase (COX)-2 at the protein levels, and iNOS, COX-2, TNF-$\alpha$ and IL-6 at the mRNA levels without an appreciable cytotoxic effect on RAW 264.7 macrophag cells. Furthermore, PCM inhibited LPS-induced nuclear factor-kappa B (NF-$\kappa$B) activation as determined by NF-$\kappa$B reporter gene assay, and this inhibition was associated with a decrease in the nuclear translocation of p65 and p50 NF-$\kappa$B. Taken together, these results suggest that PCM may play an anti-inflammatory role in LPS-stimulated RAW 264.7 macrophages through the inhibitory regulation of iNOS, COX-2, TNF-$\alpha$ and IL-6 via NF-$\kappa$B inactivation.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4363-4368
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• 2012

The epidermal differentiation complex (EDC) contains a large number of gene products which are crucial for the maturation of the human epidermis and can contribute to skin diseases, even carcinogenesis. It is generally accepted that activation of oncogenes and/or inactivation of tumor suppressor genes play pivotal roles in the process of carcinogenesis. Here, NICE-3, a novel EDC gene, was found to be up-regulated in human hepatocellular carcinoma (HCC) by quantitative real-time RT-PCR. Furthermore, overexpression of exogenous NICE-3 by recombinant plasmids could significantly promote cell proliferation, colony formation and soft agar colony formation in Focus and WRL-68 HCC cell lines. Reversely, NICE-3 silencing by RNA interference could markedly inhibit these malignant phenotypes in YY-8103 and MHCC-97H cells. Moreover, cell cycle analysis of MHCC-97H transfected with siRNA by flow cytometry showed that NICE-3 knockdown may inhibit cell growth via arrest in G0/G1 phase and hindering entry of cells into S phase. All data of our findings indicate that NICE-3 may contribute to human hepatocellular carcinoma by promoting cell proliferation.

• KSBB Journal
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• v.27 no.4
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• pp.257-262
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• 2012

The gene encoding Thermus thermophilus HJ6 laccase (Tt-laccase) was cloned, sequenced, and comprised of 1,389 nucleotides encoding a protein (462 amino acids) with a predicted molecular mass of 51,049 Da. The deduced amino acid sequence of Tt-laccase showed 99.7% and 44.3% identities to the Thermus thermophilus HB27 laccase and Synechococcus sp. RS9917 laccase, respectively. Tt-laccase gene was expressed as a fusion protein with six histidine residues in E. coli Rosetta-gami (DE3) cells, and the recombinant protein was purified to homogeneity. UV-Vis spectrum analysis revealed that the enzyme has copper atoms, a type I Cu(II) and a type III binuclear Cu(II). The optimum pH for the oxidation of guaiacol was 5.0 and the optimum temperature was $90^{\circ}C$ The half-life of heat inactivation was about 120 min at $90^{\circ}C$ The enzyme reaction was inhibited by sodium azide, L-cystein, EDTA, dithiothreitol, tropolone, and kojic acid. The enzyme oxidized various known laccase substrates, its lowest $K_m$ value being for 4-hydroxyindole, highest $k_{cat}$ value for syringaldazine, and highest $k_{cat}/K_m$ for guaiacol.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.278-281
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• 2004

The $\alpha$1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 plays a key role for the outer chain initiation of the N-linked oligosaccharides. A search for Hansenula polymorpha genes homologous to S. cerevisiae OCHI (ScOCH1) has revealed seven open reading frames (ORF100, ORF142, ORF168, ORF288, ORF379, ORF576, ORF580). All of the seven ORFs are predicted to be a type II integral membrane protein containing a transmembrane domain near the amino-terminal region and has a DXD motif, which has been found in the active site of many glycosyltransferases. Among this seven-membered OCH1 gene family of H. polymorpha, we have carried out a functional analysis of H. polymorpha ORF168 (HpOCH2) showing the highest identity to ScOCH1. Inactivation of this protein by disruption of corresponding gene resulted in several phenotypes suggestive of cell wall defects, including hypersensitivity to hygromycin B and sodium deoxycholate. The structural analysis of N-glycans synthesized in HpOCH2-disrupted strain (Hpoch2Δ) and the in vitro $\alpha$1,6-mannosyltransferase activity assay strongly indicate that HpOch2p is a key enzyme adding the first $\alpha$1,6-mannose residue on the core glycan Man$_{8}$GlcNAc$_2$. The Hpoch2Δ was further genetically engineered to synthesize a recombinant glycoprotein with the human compatible N-linked oligosaccharide, Man$_{5}$GlcNAc$_2$, by overexpression of the Aspergillus saitoi $\alpha$1,2-mannosidase with the 'HDEL” ER retention signal.gnal.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.157-167
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• 2000

A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

• International Journal of Oral Biology
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• v.32 no.2
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• pp.79-84
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• 2007

Oral squamous carcinoma (OSC) is the most common malignant neoplasm of the oral mucosa. Although the etiology of OSC is not fully understood, accumulated evidences indicate that the activation of proto-oncogenes and the inactivation of tumor suppressor genes underlie the disease development. An OSC cell line, YD-9 was newly established and characterized. However, the mutational analysis of p53 gene was not performed. Thus, in this study, the presence of mutation in the p53 gene was examined by amplification of exon-4 to -8 and subsequent DNA sequencing. Two point mutations were found in exon-4 and -6: A to G, resulting in amino acid change Tyr to Cys in exon-4, and C to G, resulting in amino acid change Gly to Arg in exon-6, respectively. Any mutation was not found in the exon-5, -7 and -8. The presented results would contribute to basic research to understand the biological mechanism of OSC using YD-9 cells.