A binding site for the cyclic adenosine 3',5'-monophosphate-response element-binding protein as a regulatory element in the grp78 promoter.

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.

Observation of the C2H2-N2O van der Waals complex in the overtone range using CW-CRDS

A slit nozzle supersonic expansion containing C2H2 (246 sccm) and N2O (355 sccm) seeded into Ar (1260 sccm) is investigated using CW cavity ring-down spectroscopy, in the 1.5 μm range. The C2H2-N2O van der Waals complex is observed around the 2CH acetylenic band. Despite strong perturbations, 117 b-type lines are assigned. Their combined fit with published microwave data leads to new upper state and improved lower state rotational constants. The Lorentzian width of the assigned line profiles sets the mean lifetime to 1.6 ns. The rotational temperature is estimated to be 15 K from the comparison between observed and simulated spectra. © 2008 Elsevier B.V. All rights reserved.