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-kappaB, 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-kappaB 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 GO6976, 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.

We identified the promoter region of the human GD3 synthase (hST8Sia I) gene to elucidate the mechanism underlying the regulation of hST8Sia I expression in human glioblastoma cells. The 5 0 -rapid amplification of cDNA end using mRNA prepared from U-87MG cells revealed the presence of transcription start site of hST8Sia I gene, and the 5 0 -terminal analysis of its product showed that transcription started from 648 nucleotides upstream of the translational initiation site. Functional analysis of the 5 0 -flanking region of the hST8Sia I gene by transient expression method revealed that the region from 2638 to 2498 is important for transcriptional activity of the hST8Sia I gene in U-87MG and T98G cells. This region lacks apparent TATA and CAAT boxes, but contains putative binding sites for transcription factors AREB6 and Elk-1. Sitedirected mutagenesis and transient transfection assays demonstrated that both AREB6 and Elk-1 elements in this region were required for the promoter activity in U-87MG and T98G cells. These results indicated that both AREB6 and Elk-1 might play an essential role in the transcriptional activity of hST8Sia I gene essential for GD3 synthesis in human glioblastoma cells.

An integrated microdevice of a reverse transcription-polymerase chain reaction (RT-PCR) reactor and an immunochromatographic strip was constructed for colorimetric detection of gene expression of influenza A virus subtype H1N1. An RT-PCR cocktail, which included Texas Red-labeled primers, dNTP including biotin-labeled dUTP, and RNA templates of influenza A H1N1 virus, was filled in the PCR chamber through the micropump, and the RT-PCR was performed to amplify the target H1 gene (102 bp). The resultant amplicons bearing biotin moieties and Texas Red haptens were subsequently eluted to the immunochromatographic strip, in which they were first conjugated with the gold nanoparticle labeled anti-hapten antibody in the conjugation pad, and then captured on the streptavidin coated test line through the biotin-streptavidin interaction. By observing a violet color in the test line which was derived from the gold nanoparticle, we confirmed the H1N1 target virus. The entire process on the integrated microdevice consisting of a micropump, a 2 μL PCR chamber, and an immunochromatographic strip was carried out on the portable genetic analyzer within 2.5h, enabling on-site colorimetric pathogen identification with detection sensitivity of 14.1 pg RNA templates.

Key wordsvalproic acid; human GM3 synthase; human n e u r o b l a s t o m a ; C R E B ; t r a n s c r i p t i o n a l regulation 1 This work was supported by a grant (M1061 9010001-08N1901-00110) from the Korea Science and Engineering Foundation (KOSEF). 6 Correspondence to Prof Young-choon LEE. 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 immunofluorescent 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, sitedirected 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.

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