Kyoung-Sook Kim

Effect of dietary Platycodon grandiflorum on the improvement of insulin resistance in obese Zucker rats.

The effect of dietary Platycodon grandiflorum on the improvement of insulin resistance and lipid profile was investigated in lean (Fa/-) and obese (fa/fa) Zucker rats, a model for noninsulin dependent diabetes mellitus. Dietary Platycodon grandiflorum feeding for 4 weeks resulted in a significant decrease in the concentration of plasma triglyceride in both lean and obese Zucker rats. Furthermore, dietary Platycodon grandiflorum markedly decreased both plasma cholesterol and fasting plasma insulin levels, and significantly decreased the postprandial glucose level at 30 min during oral glucose tolerance test in obese Zucker rats. Although there was no statistical significance, the crude glucose transporter 4 protein level of obese rats fed Platycodon grandiflorum tended to increase when compared with that of obese control rats. Therefore, the present results suggested that dietary Platycodon grandiflorum may be useful in prevention and improvement of metabolic disorders characterized by hyperinsulinemia states such as noninsulin dependent diabetes mellitus, syndrome X, and coronary artery disease.

Isolation and characterization of Pseudomonas otitidis WL-13 and its capacity to decolorize triphenylmethane dyes

Pseudomonas otitidis WL-13, which has a high capacity to decolorize triphenylmethane dyes, was isolated from activated sludge obtained from a wastewater treatment plant of a dyeing industry. This strain exhibited a remarkable color-removal capability when tested against several triphenylmethane dyes under both shaking and static conditions at high concentrations of dyes. More than 95% of Malachite Green and Brilliant Green was removed within 12 h at 500 micromol/L dye concentration under shaking conditions. Crystal Violet lost about 13% of its color under the same conditions tested. The rate of decolorization increased when the M9 medium was supplemented with yeast extract. The optimum pH and temperature for color removal were 7-9 and 35-40 degrees C, respectively. The observed changes in the visible spectra and the inspection of bacterial growth indicated the color-removal by the adsorption of dye to the cells during incubation with strains.

Isolation and characterization of the promoter region of the human GM3 synthase gene

GM3 synthase, which transfers CMP-NeuAc with an alpha2,3-linkage to a galactose residue of lactosylceramide, plays a key role in the biosynthesis of all complex gangliosides. The expression of this gene is highly restricted in both human fetal and adult tissues. To elucidate the mechanisms that regulate the tissue-specific expression of the human GM3 synthase (hST3Gal V) gene, we have isolated and characterized its 5-flanking region. Potential transcriptional start site was determined by CapSite hunting. Sequence analysis of the 5-flanking region revealed that hST3Gal V promoter lacked canonical TATA and CAAT boxes but contained several putative binding sites for transcription factors AP4, MZF1, SP1, ATF/CREB, NFY, IK2 and LYF1, etc. Functional analysis of the 5-flanking region of the hST3Gal V gene by transient expression method revealed that the -177 to -83 region is important for transcriptional activity of the hST3Gal V gene in SK-N-MC and HepG2 cells. The present results also suggested that both positive and negative regulatory elements are present in this TATA-less promoter of the hST3Gal V gene.

Cloning, expression in Escherichia coli, and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of Klebsiella sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS (2,2-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol), respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Valproic acid induces transcriptional activation of human GD3 synthase (hST8Sia I) in SK-N-BE(2)-C human neuroblastoma cells

In this study, we have shown the transcriptional regulation of the human GD3 synthase (hST8Sia I) induced by valproic acid (VPA) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the regulation of hST8Sia I gene expression in VPA-stimulated SK-N-BE(2)-C cells, we characterized the promoter region of the hST8Sia I gene. Functional analysis of the 5′-flanking region of the hST8Sia I gene by the transient expression method showed that the −1146 to −646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-κB, functions as the VPA-inducible promoter of hST8Sia I in SK-N-BE(2)-C cells. Site-directed mutagenesis and electrophoretic mobility shift assay indicated that the NF-κB binding site at −731 to −722 was crucial for the VPA-induced expression of hST8Sia I in SK-N-BE(2)-C cells. In addition, the transcriptional activity of hST8Sia I induced by VPA in SK-N-BE(2)-C cells was strongly inhibited by SP600125, which is a c-Jun N-terminal kinase (JNK) inhibitor, and GÖ6976, which is a protein kinase C (PKC) inhibitor, as determined by RT-PCR (reverse transcription-polymerase chain reaction) and luciferase assays. These results suggest that VPA markedly modulated transcriptional regulation of hST8Sia I gene expression through PKC/JNK signal pathways in SK-N-BE(2)-C cells.

Plasma membrane localization of soybean matrix metalloproteinase differentially induced by senescence and abiotic stress.

We isolated and characterized a stress-inducible gene, designated as Slti114, encoding matrix metalloproteinase (MMP) in soybean. The derived amino acid sequences of Slti114 show the 69 % homology with MMP2 from Glycine max (AAL27029). The size of the full-length cDNA of Slti114 is 1194 bp with open reading frame comprised of 394 amino acids. RNA expression of Slti114 was induced by low temperature or wounding. During early stage, Slti114 RNA level was extremely high, but Slti114 RNA was not detectable just after cotyledons became yellowish. Green fluorescent protein fusion expression system confirmed that Slti114-smGFP and H+-ATPase-RFP were co-localized to the plasma membrane. Purified glutathione-S-transferase (GST)-Slti114 protein was shown to digest myelin basic protein (MBP) in vitro, but not gelatin. This report provides strong evidence that plasma membrane MMP, Slti114 protein may play a critical role during abiotic stress and senescence in plant.

Anti-inflammatory effects of 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone via NF-κB inactivation in lipopolysaccharide-stimulated RAW 264.7 macrophage

The anti-inflammatory mechanism of 5-hydroxy-3,6,7,8,3,4-hexamethoxyflavone (5HHMF), a polyhydroxyflavone isolated from the marine algae Hizikia fusiforme, was investigated in RAW 264.7 murine macrophage cells. Western blot and reverse transcriptase PCR analyses indicated that adding 5HHMF to cultured cells significantly reduced the production of nitric oxide and prostaglandin E2 and downregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In addition, 5HHMF inhibited the release of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β, and decreased the transcriptional levels. In particular, 5HHMF significantly inhibited the LPS-induced nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus, which was associated with the abrogation of inhibitory IκBα degradation and subsequent decreases in nuclear p65 levels. In conclusion, these results suggested that the anti-inflammatory activities of 5HHMF may be attributed to the inhibition of iNOS, COX-2 and cytokine expression by attenuating NF-κB activation via IκBα degradation in macrophages.

Molecular characterization of soybean ribosomal protein S13 targeted to the nucleus

We isolated the full-length genomic clone of STLI25 encoding RPS13 (ribosomal protein S13) in soybean. Genomic DNA structure of SLTI25 is similar to that of Arabidopsis ribosomal protein S13. RNA expression of SLTI25 was induced by salt, ABA, or wounding stress, but reduced by dehydration stress. To determine the subcellular localization of the gene product fused to GFP, we were able to confirm that SLTI25-GFP was restricted to the nucleus. By domain swapping analysis, it was shown that C-terminal region of SLTI25 with a putative nucleus localization signal was necessary and sufficient for nucleus targeting of the fusion protein in plants. This new findings provide an evidence that SLTI25 is targeted to the nucleus for the ribosome subunit assembly in plants.

Valproic acid-mediated transcriptional regulation of human GM3 synthase (hST3Gal V) in SK-N-BE(2)-C human neuroblastoma cells

AbstractAim:To investigate whether valproic acid (VPA) modulates human GM3 synthase (hST3Gal V) mRNA expression, as a part of ganglioside GM3 biosynthesis, in human neuroblastoma cells.Methods:Using RT-PCR and immunofluo-rescent confocal microscopy, we examined hST3Gal V mRNA and GM3 levels during VPA-induced differentiation of human neuroblastoma SK-N-BE(2)-C cells. We characterized the VPA-inducible promoter region within the hST3-Gal V gene using luciferase constructs carrying 5′-deletions of the hST3Gal V promoter.Results:RT-PCR indicated that VPA-mediated hST3Gal V induction is transcriptionally regulated. Functional analysis of the 5′-flanking region of the hST3Gal V gene demonstrated that the -177 to -83 region, which contains a cAMP-responsive element (CRE) at -143, functions as the VPA-inducible promoter by actively binding CRE binding protein (CREB). In addition, site-directed mutagenesis and electrophoretic mobility shift assay indicated that the CRE at -143 is crucial for the VPA-induced expression of hST3Gal V in SK-N-BE(2)-C cells.Conclusion:Our results isolated the core promoter region in the hST3Gal V promoter, a CRE at -143, and demonstrated that it is essential for transcriptional activation of hST3Gal V in VPA-induced SK-N-BE(2)-C cells. Subsequent CREB binding to this CRE mediates VPA-dependent upregulation of hST3Gal V gene expression.

Transcriptional activation of pig Galβ1,3GalNAc α2,3-sialyltransferase (pST3Gal I) gene by TGF-β1 in porcine kidney PK-15 cells

In this study we investigated for the first time the transcriptional regulation of pig Galβ1,3GalNAc α2,3-sialyltransferase (pST3Gal I) in response to TGF-β1 in porcine kidney PK-15 cells. The pST3Gal I gene was found to span about 90kb and to be composed of 8 exons including 2 exons in the 5-untranslated region. RT-PCR analysis indicated that the induction of pST3Gal I by TGF-β1 is regulated at the transcriptional level. Functional analysis of the 5-flanking region of the pST3Gal I gene revealed the -1257 to -976 region functions as the TGF-β1-inducible promoter and that the Smad-binding site at -1020 is crucial for TGF-β1-induced expression of pST3Gal I in PK-15 cells. In addition, the transcriptional activity of pST3Gal I induced by TGF-β1 in PK-15 cells was strongly inhibited by SIS3, which is a specific Smad-3 inhibitor. In summary, our results identified the core promoter region in the pST3Gal I promoter and demonstrated that Smad-3 binding to the Samd-3 binding site at -1020 is essential for transcriptional activation of pST3Gal I in TGF-β1-induced PK-15 cells.