• BMB Reports
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• v.33 no.3
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• pp.195-201
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• 2000

A human gene has been reported that may encode the enzyme acetohydroxyacid synthase. Previously this enzyme was thought to be absent from animals although it is present in plants and many microorganisms. In plants, this enzyme is the target of a number of commercial herbicides and the use of these compounds may need to be reassessed if the human enzyme exists and proves to be susceptible to inhibition. Here we report the construction of several plasmid vectors containing the cDNA sequence for this protein, and their expression in Escherichia coli. High levels of expression were observed, but most of the protein proved to be insoluble. The small amounts of soluble protein contained little or no acetohydroxyacid synthase activity. Attempts to refold the insoluble protein were successful insofar as the protein became soluble. However, the refolded protein did not gain any acetohydroxyacid synthase activity. In vivo complementation tests of an E. coli mutant produced no evidence that the protein is active. Incorrect folding, or the lack of another subunit, may explain the data but we favor the interpretation that this gene does not encode an acetohydroxyacid synthase.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.35-39
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• 2001

The Integration Host factor (IHF) of Escherichia coli is a small, basic protein that is required for a variety of functions including site-specific recombination, transposition, gene regulation, plasmid replication, and DNA packaging. It ,is composed of two subunits that are encoded by the ihfA ($\alpha$-subunit) and ihjB ($\beta$-subunit) genes. IHF binding sites are composed of three elements called the WATCAR, TTG, and poly (dAT) elements. We have characterized IHF binding to the H site of bacteriophage λ. We have isolated suppressors that bind to altered H' sites using a challenge phage selection. Two different suppressors were isolated that changed the adjacent $\alpha$P64 and $\alpha$K65 residues. The suppressors recognized both the wild-type site and a site with a change in the WATCAR element. Three suppressors were isolated at $\beta$-E44. These suppressors bound the wild-type and a mutant site with a T：A to A：T change (H44A) in the middle of the TIR element. Site-directed mutagenesis was used to make several additional changes at $\beta$E44. The wild-type and $\beta$E44D mutant could not bind the wild-type site but were able to bind the H44A mutant site. Other mutants with neutral, polar, or a positive charge at $\beta$E44 were able to repress both the wild-type and H44A sites. Examination of the IHF crystal structure suggests that the ability of the wild-type and $\beta$E44D proteins to discriminate between the T：A and A：T basepairs is due to indirect interactions. The $\beta$-E44 residue does not contact the DNA directly. It imposes binding specificity indirectly by interactions with residues that contact the DNA. Details of the proposed interactions are discussed.

• Parasites, Hosts and Diseases
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• v.47 no.2
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• pp.117-124
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• 2009

Toxoplasma gondii provokes rapid and sustained nuclear translocation of the signal transducer and activator of transcription 6 (STAT6) in HeLa cells. We observed activation of STAT6 as early as 2hr after infection with T. gondii by the nuclear translocation of fluorescence expressed from exogenously transfected pDsRed2-STAT6 plasmid and by the detection of phosphotyrosine-STAT6 in Western blot. STAT6 activation occurred only by infection with live tachyzoites but not by co-culture with killed tachyzoites or soluble T. gondii extracts. STAT6 phosphorylation was inhibited by small interfering RNA of STAT6 (siSTAT6). In view of the fact that STAT6 is a central mediator of IL-4 induced gene expression, activation of STAT6 by T. gondii infection resembles that infected host cells has been stimulated by IL-4 treatment. STAT1 was affected to increase the transcription and expression by the treatment of siSTAT6. STAT6 activation was not affected by any excess SOCS's whereas that with IL-4 was inhibited by SOCS-1 and SOCS-3. T. gondii infection induced Eotaxin-3 gene expression which was reduced by $IFN-{\gamma}$. These results demonstrate that T. gondii exploits host STAT6 to take away various harmful reactions by $IFN-{\gamma}$. This shows, for the first time, IL-4-like action by T. gondii infection modulates microbicidal action by $IFN-{\gamma}$ in infected cells.

• Journal of Microbiology
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• v.34 no.1
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• pp.37-37
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• 1996

The internal regions of nuclear small subunit rRNA from 6 plaeurotus species and 5 Pleurotus ostreatus strains were amplified by PCR and sequenced. The DNA sequences of 8 Pleurotus strains (P. ostreatus NFFA2, NFFA4501, NFFA4001, KFFA4001, KFCC11635, P florida, P. florida, P. sajor-cuju, P. pulmonarius, and P. spodoleucus) were idential, but P. cornucopiae differed from them in two bases out of 605 bases. However, p[hylogenetic analysis of the sequences by DNA-distance matrix and UPGMA methods showed that P. ostreatus NFFA2m1 and NFFA2m2, known as mutants of P. ostreatus NFFA2, belonged to anther group of Basidiomycotina, which is close to the genus Auricularia. The difference of the SSU rDNA sequences of P. cornucopiae from other Pleurotus species tested corresponds to the difference of mitochondrial plasmid type present in Pleurotus species as observed by Kim et al. (1993, Korean J. Microbiol. 31, 141-147).ishement of silencing at the HMR/hsp82 locus can occur in G1-arrested cells. Cell cycle arrest at G1 phase was achieved by treatment of early log a cell cultures with .alpha.-factor mating pheromone, which induces G1 arrest. The result suggests that passage through S phase (and therefore DNA replication) is nor required for re-establishing silencer-mediated repression at the HMNRa/HSP82 locus. Finally, to test whether de nono protein synthesis is required for re-establishment of silencer-mediated repression, cells were pretreated with cycloheximide (500 /.mu.g/ml) 120 min. It was apparent that inhibiting protein synthesis delays, but does not prevent, re-establishment of silencer-mediated repression. Altogether, these results indicate that re-establishment of silencer-mediated repression is not dependent on the DNA replication and has no requirement for protein synthesis.

• BMB Reports
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• v.52 no.12
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• pp.706-711
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• 2019

Cisplatin (Cis-DDP) is one of the most widely used anti-cancer drugs. It is applicable to many types of cancer, including lung, bladder, and breast cancer. However, its use is now limited because of drug resistance. p90 ribosomal S6 kinase (p90RSK) is one of the downstream effectors in the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathway and high expression of p90RSK is observed in human breast cancer tissues. Therefore, we investigated the role of p90RSK in the Cis-DDP resistance-related signaling pathway and epithelial-mesenchymal transition (EMT) in breast cancer cells. First, we discovered that MDA-MB-231 cells exhibited more Cis-DDP resistance than other breast cancer cells, including MCF-7 and BT549 cells. Cis-DDP increased p90RSK activation, whereas the inactivation of p90RSK using a small interfering RNA (siRNA) or dominant-negative kinase mutant plasmid overexpression significantly reduced Cis-DDP-induced cell proliferation and migration via the inhibition of matrix metallopeptidase (MMP)2 and MMP9 in MDA-MB-231 cells. In addition, p90RSK activation was involved in EMT via the upregulation of mRNA expression, including that of Snail, Twist, ZEB1, N-cadherin, and vimentin. We also investigated NF-κB, the upstream regulator of EMT markers, and discovered that Cis-DDP treatment led to NF-κB translocation in the nucleus as well as its promoter activity. Our results suggest that targeting p90RSK would be a good strategy to increase Cis-DDP sensitivity in triple-negative breast cancers.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.15-21
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• 2013

We previously observed that Bacillus subtilis spores from sspE mutants presented a lower germination capacity in media containing high salt concentrations (0.9M NaCl). This deficiency was attributed to the absence of SASP-E (gamma-type small-acid-soluble protein), rich in osmocompatible amino acids released by degradation. Herein we observed that, in addition, this mutant spore presented a reduced capacity to use L-alanine as germinant (L-ala pathway), required longer times to germinate in calcium dipicolinate ($Ca^{2+}$-DPA), but germinated well in asparagine, glucose, fructose, and potassium chloride (AGFK pathway). Moreover, mild sonic treatment of mutant spores partially recovered their germination capacity in L-ala. Spore qualities were also altered, since sporulating colonies from the sspE mutant showed a pale brownish color, a higher adherence to agar plates, and lower autofluorescence, properties related to their spore coat content. Furthermore, biochemical analysis showed a reduced partition in hexadecane and a higher content of $Ca^{2+}$-DPA when compared with its isogenic wild-type control. Coat protein preparations showed a different electrophoretic pattern, in particular when detected with antibodies against CotG and CotE. The complementation with a wild-type sspE gene in a plasmid allowed for recovering the wild-type coat phenotype. This is the first report of a direct involvement of SASP-E in the spore coat assembly during the differentiation program of sporulation.

• Korean Journal of Microbiology
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• v.40 no.1
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• pp.7-11
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• 2004

eIF5B is a translation initiation factor that delivers Met-$tRNA^{Met}$ to AUG start codon and subsequently joins the small and large ribosomes. In order to study the function of eIF5B encoded by FUN12, we constructed FUN12 which lacked 5' end of its sequence. We found that this construct lacking almost all of its promoter in pRS plasmid partially complemented slow growth phenotype of fun12 deletion strain. Interestingly, this construct expressed N-terminally truncated eIF5B and its expression level was about 5% of that of wild type eIF5B. Low amount of the eIF5B expressed additionally in fun12 deletion strain played a direct role as a limiting factor for its growth. This limiting factor eIF5B in those strains also modulates activities of overall translation in vitro.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• Journal of Ginseng Research
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• v.46 no.2
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• pp.248-254
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• 2022

Background: Zinc homeostasis is essential for human health and is regulated by several zinc transporters including ZIP and ZnT. ZIP4 is expressed in the small intestine and is important for zinc absorption from the diet. We investigated in the present study the effects of Panax ginseng (P. ginseng) extract on modulating Zip4 expression and cellular zinc levels in mouse Hepa cells. Methods: Hepa cells were transfected with a luciferase reporter plasmid that contains metal-responsive elements, incubated with P. ginseng extract, and luciferase activity was measured. Using ⁶⁵ZnCl₂, zinc uptake in P. ginseng-treated cells was measured. The expression of Zip4 mRNA and protein in Hepa cells was also investigated. Finally, using a luciferase reporter assay system, the effects of several ginsenosides were monitored. Results: The luciferase activity in cells incubated with P. ginseng extract was significantly higher than that of control cells cultured in normal medium. Hepa cells treated with P. ginseng extract exhibited higher zinc uptake. P. ginseng extract induced Zip4 mRNA expression, which resulted in an enhancement of Zip4 protein expression. Furthermore, some ginsenosides, such as ginsenoside Rc and Re, enhanced luciferase activity driven by intracellular zinc levels. Conclusion: P. ginseng extract induced Zip4 expression at the mRNA and protein level and resulted in higher zinc uptake in Hepa cells. Some ginsenosides facilitated zinc influx. On the basis of these results, we suggest a novel effect of P. ginseng on Zip4-mediated zinc influx, which may provide a new strategy for preventing zinc deficiency.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.325-330
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• 2007

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell and expression of N-terminal domain consisted of 1-498 amino acids (N-Rne) is sufficient to support normal cellular growth. By utilizing these properties of RNase E, we developed a genetic system to screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that lead to various phenotypes. Using this system, we identified three kinds of mutants. A mutant N-Rne containing amino acid substitution in the S1 domain (I6T) of the protein was not able to support survival of E. coli cells, and another mutant N-Rne with amino acid substitution at the position 488 (R488C) in the small domain enabled N-Rne to have an elevated ribonucleolytic activity, while amino acid substitution in the DNase I domain (N305D) only enabled N-Rne to support survival of E. roli cells when the mutant N-Rne was over-expressed. Analysis of copy number of ColEl-type plasmid revealed that effects of amino acid substitution on the ability of N-Rne to support cellular growth stemmed from their differential effects on the ribonucleolytic activity of N-Rne in the cell. These results imply that the genetic system developed in this study can be used to isolate mutant RNase E with various phenotypes, which would help to unveil a functional role of each subdomain of the protein in the regulation of RNA stability in E. coli.