• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1527-1535
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• 2009

Polyphosphate (polyP) plays diverse physiological functions in prokaryotes and eukaryotes, but most of their detailed mechanisms are still obscure. Here, we show that deletion of polyphosphate kinase (PPK), the principal enzyme responsible for synthesis of polyP, resulted in augmented expression of cAMP receptor protein (CRP) and rpoS and lowered $H_2O_2$ sensitivity in Salmonella Typhimurium ATCC14028. The binding of cAMP-CRP complex to rpoS promoter and further stimulation of its transcription were proved through electrophoretic mobility shift assay, lacZ fusion, and exogenous cAMP addition, respectively. The rpoS expression increased in cpdA (cAMP phosphodiesterase coding gene) mutant, further suggesting that cAMP-CRP upregulated rpoS expression. These results demonstrate that PPK affects oxidative stress response by modulating crp and rpoS expression in S. Typhimurium.

• Molecules and Cells
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• v.20 no.3
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• pp.364-370
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• 2005

We show that sulforaphane inhibits osteoclastogenesis in the presence of macrophage colony-stimulating factor (M-CSF) and receptor for activation of nuclear factor-${\kappa}B$ ligand (RANKL) in osteoclast (OC) precursors. Sulforaphane, an aliphatic isothiocyanate, is a known cancer chemo-preventative agent with anti-oxidative properties. Nuclear factor-${\kappa}B$ (NF-${\kappa}B$) is a critical transcription factor in RANKL-induced osteoclastogenesis, and electrophoretic mobility shift assays (EMSAs) and assay of NF-${\kappa}B$-mediated secreted alkaline phosphatase (SEAP) revealed that sulforaphane selectively inhibited NF-${\kappa}B$ activation induced by RANKL. Inhibition may involve interaction of sulforaphane with thiol groups, since it was prevented by reducing agents.

• BMB Reports
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• v.30 no.3
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• pp.205-210
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• 1997

p53 tumor suppressor plays an important role in the regulation of cellular proliferation. To identify proteins regulating the expression of p53 in rat liver, we analyzed p53 promoter by electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay. We found that a protein binds the sequence CACGTG, bHLH consensus sequence in rat p53 promoter. Southwestern blotting analysis with oligonucleotides containing this sequence shows that the molecular weight of the protein is 100 kDa. This size is not compatible with the bHLH family such as USF or c-Myc/Max which is known to regulate the expression of the human and mouse p53 gene. Therefore this 100 kDa protein may be a new protein regulating basal transcription of rat p53. We purified this 100 kDa protein through sequence-specific DNA affinity chromatogaphy.

• Development and Reproduction
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• v.16 no.4
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• pp.379-384
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• 2012

Lhx8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian folliculogenesis. However, Lhx8 DNA binding sequences are not characterized yet. We aimed to identify and characterize a cis-acting sequence of germ-cell specific transcriptional factor, Lhx8. To identify Lhx8 DNA binding element, Cyclic Amplification of Sequence Target (CAST) Analysis was performed. Electrophoretic Mobility Shift Assay (EMSA) was processed for the binding specificity of Lhx8. Luciferase assay was for the transcriptional activity of Lhx8 through identified DNA binding site. We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE). In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element. These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

• The Journal of Korean Medicine
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• v.21 no.4
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• pp.64-72
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• 2000

Objectives : This study was designed to investigate the neuroprotective effect of Hwansodan(Huanshao-dan) on the apoptosis induced by withdrawal of neurotrophic support. Methods : PCl2 pheochromocytoma cells have been used extensively as a model for studying the cellular and molecular effects of neuronal cells. The viability of cells was measured by MTT assay. We used DNA fragmentation and caspase 3-like protease activation assay. Results : The water extract of Hwansodan(Huanshao-dan) significantly showed protective effects on serum and glucose deprivation-induced apoptotic death. Hwansodan(Huanshao-dan) also prevents DNA fragmentation and caspase 3-like protease activation, representing typical neuronal apoptotic phenomena in PCl2 pheochromocytoma cells and induces tyrosine phosphorylation of proteins around 44 kDa, which was identified as ERK1 with electrophoretic gel mobility shift by Western blot. In addition, MAPK kinase(MEK) inhibitor PD98059 and Ras inactivator, ${\alpha}-hydroxyfarnesylphosphonic$ acid attenuated the neuroprotective effects of Hwansodan(Huanshao-dan) in serum-deprived PCl2 cells. Conclusions : These results indicate that Ras/MEK/ERK signaling pathway plays a key role in neuroprotective effects of Hwansodan(Huanshao-dan) in serum-deprived PCl2 cells. Taken together, we suggest the possibility that Hwansodan(Huanshao-dan) might provide a neurotrophic-like activity in PCl2 cells.

• Molecules and Cells
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• v.28 no.3
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• pp.189-194
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• 2009

The modulating effect of IGF-I on the regulation of AR gene expression and activation in skeletal muscle cells remains poorly understood. In this study, the effects of IGF-I treatment on AR induction and activation in the absence of AR ligands were examined. Differentiating C2C12 cells were treated with different concentrations (0-250 ng/ml) of IGF-I or for various periods of time (0-60 min) of 250 ng/ml IGF-I. Treatment of C2C12 cells with IGF-I resulted in a dose- and time-dependent increase in total AR and phosphorylated AR (Ser 213). IGF-I treatment also led to significantly increased AR mRNA expression when compared with the control. The levels of skeletal ${\alpha}-actin$ and myogenin mRNA, known target genes of AR, were also significantly upregulated after 5 or 10 min of treatment with IGF-I. Confocal images revealed that IGF-I stimulated nuclear localization of AR in the absence of ligands. In addition, an electrophoretic mobility shift assay indicated that IGF-I stimulated the AR DNA binding activity in a time-dependent manner. The present results suggest that IGF-I stimulates the expression and activation of AR by ligand-independent mechanism in differentiating C2C12 mouse skeletal muscle cells.

• Toxicological Research
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• v.22 no.2
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• pp.103-107
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• 2006

We demonstrate that paclitaxel, an antitumor agent derived from yew tree, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. Previously, paclitaxel has been known to induce iNOS gene expression in macrophages. However, in this report we described that the pre-treatment of macrophages with paclitaxel ($0.1{\mu}M$) for 8 h inhibited LPS-induced iNOS gene expression. Pretreatment of RAW 264.7 cells with paclitaxel significantly inhibited LPS-stimulated nitric oxide (NO) production. Western immunoblot of iNOS and RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression in RAW 264.7 cells. Immunocytochemical staining of iNOS further confirmed that pretreatment of macrophages with paclitaxel inhibited macrophage activation. Electrophoretic mobility shift assay showed that paclitaxel inhibited $NF-_{\kappa}/Rel$ DNA binding. Collectively, these series of experiments indicate that paclitaxel inhibits iNOS gene expression by blocking $NF-_{\kappa}B/Rel$ activation.

• IMMUNE NETWORK
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• v.4 no.2
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• pp.116-122
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• 2004

Background: LIGHT (TNFSF14) is a member of tumor necrosis factor superfamily and is the ligand for TR2 (TNFRSF14/HVEM). LIGHT is known to have proinflammatory roles in atherosclerosis. Methods: To find out the expression pattern of LIGHT in atherosclerotic plaques, immunohistochemical analysis was performed on human carotid atherosclerotic plaque specimens. LIGHT induced atherogenic events using human monocytic cell line THP-1 were also investigated. Results: Immunohistochemical analysis revealed expression of LIGHT and TR2 in foam cell rich regions in the atherosclerotic plaques. Double immunohistochemical analysis further confirmed the expression of LIGHT in foam cells. Stimulation of THP-1 cells, which express TR2, with either recombinant LIGHT or immobilized anti-TR2 monoclonal antibody induced interleukin-8 and matrix metalloproteinase(MMP)-9. Electrophoretic mobility shift assay demonstrated that LIGHT induces nuclear localization of transcription factor, nuclear factor $(NF)-{\kappa}B$. LIGHT induced activation of MMP-9 is mediated by $NF-{\kappa}B$, since treatment of THP-1 cells with the $NF-{\kappa}B$ inhibitor PDTC (pyrrolidine dithiocarbamate) completely blocked the activation of MMP-9. Conclusion: These data indicate that LIGHT is expressed in foam cells in atherosclerotic plaques and is involved in atherogenesis through activation of pro-atherogenic cytokine IL-8 and destabilization of plaque by inducing matrix degrading enzyme.

• Parasites, Hosts and Diseases
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• v.42 no.2
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• pp.57-60
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• 2004

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

• Journal of Life Science
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• v.6 no.4
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• pp.234-240
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• 1996

A double-stranded RNA binding factor (RBF), characterized as an inhibitor of PKR kinase in our previous study, was evaluated for its RNA binding specificities by RNA gel electrophoretic mobility shift analysis and membrane filter binding assay, RBF displayed affinities for a broad range of RNAs including viral RNAs and synthetic RNAs consiting of stem and loop structures. GC-rich RNA stem helices as short as 11 bp are suggested to represent the minimal binding motif for RBF. RBF binding to all the natural RNAs tested was reversible by poly(I): poly(C) addition, but E. coli 5S RNA was inefficient.