Application of post-column reaction gas chromatography with a single reference gas for offshore air and gas seeped from the seafloor samples.

Ambient air commonly contains carbon dioxide at concentrations greater than 400 µmol mol-1 and methane at ~ 2000 nmol mol-1; non-methane hydrocarbons are also widespread in the atmosphere at much lower concentrations. For quantification of various carbon-containing compounds in typical analytical instrument, corresponding number of reference materials are required. Therefore, the development of a method that uses a single reference material applicable to air monitoring is desired. Here, we examined a post-column reaction system combined with a gas chromatograph equipped with a flame ionization detector (FID), which involves oxidation and reduction processes after separation. To determine various carbon-containing gases by post-column reaction gas chromatography with FID (GC-r-FID) using a single reference, it is necessary to confirm a good linearity of the response with carbon concentrations originating from various carbon-containing gases. When mixtures of carbon-containing gases at three different concentrations and the calibration curve of the FID response with the concentration converted into methane were used, a single linear calibration curve (correlation coefficient > 0.9999, 18 points) was obtained over four orders of magnitudes (to ~ 5000 µmol mol-1 as methane). The applicability of GC-r-FID was confirmed by determining carbon-containing gases in air and gas seeped from the seafloor samples. Because the results were comparable to those obtained by conventional GC-FID and GC-thermal conductivity detector, typically GC-r-FID with a single reference gas should be suitable for air monitoring.

Identification of PITX1 as a TERT suppressor gene located on human chromosome 5.

Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37-47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.

ST2 gene expression is proliferation-dependent and its ligand, IL-33, induces inflammatory reaction in endothelial cells.

ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.

ST2 suppresses IL-6 production via the inhibition of IkappaB degradation induced by the LPS signal in THP-1 cells.

LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-kappaB to the IL-6 promoter. Furthermore, the degradation of IkappaB in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated that ST2 negatively regulates LPS-induced IL-6 production via the inhibition of IkappaB degradation in THP-1 cells.

T-helper type 2 cell-specific expression of the ST2 gene is regulated by transcription factor GATA-3.

ST2 is a member of the interleukin-1 receptor family and is expressed in type-2 T helper (Th2) cells. Here, we have studied the molecular mechanism responsible for the transcriptional regulation of the ST2 gene in Th2 cells using a mouse thymoma cell line, EL-4. The ST2 gene has distal and proximal promoters. ST2 mRNA was produced from the distal promoter in EL-4 cells stimulated with both phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt2cAMP). The region of approximately 100 bp upstream of transcription start site, containing two GATA consensus sites, was indispensable for the activation of the distal promoter in reporter gene analysis. An electrophoretic mobility shift assay showed that transcription factor GATA-3 bound one of the GATA consensus sites (from -84 to -79) with nuclear extracts from PMA plus Bt2cAMP-stimulated EL-4 cells. The overexpression of GATA-3 enhanced the activity of the distal promoter. On the other hand, mutations of the GATA consensus site canceled out the enhancement by GATA-3. These data suggest that GATA-3 is an important transcription factor for the expression of the ST2 gene in Th2 cells.