Modulation of JunD.AP-1 DNA binding activity by AP-1-associated factor 1 (AF-1)

AP-1-associated factor 1 (AF-1), is a novel protein complex that dramatically enhances the assembly of JunD-containing dimers onto AP-1 consensus sites. We describe the partial purification of AF-1 from nuclear extracts of the T-cell line MLA 144 by ionic, hydrophobic and gel filtration chromatography. AF-1 is a DNA-binding protein composed of low molecular mass polypeptides of 7-17 kDa that exists in solution as a 34-kDa complex. JunD interactions with DNA are accelerated in the presence of AF-1 through the formation of a true tri-molecular complex with JunD dimers and DNA that assembles much more rapidly on DNA than JunD alone. DNA binding analysis of AF-1 interaction with JunD.AP-1 and DNA shows that AF-1 increases the DNA binding affinity of JunD for AP-1 sites over 100-fold. DNA cleavage footprint analysis of isolated AF-1.JunD DNA complexes shows that the ternary complex makes nearly twice as many contacts with DNA than JunD dimers alone. AF-1 interacts readily, but differentially with Jun homodimers and Jun.Fos heterodimers. These findings distinguish AF-1 as a significant protein-specific modulator of AP-1.JunD in T-cells.     

Localization of putative elements of 5''-region of human tyrosine aminotransferase gene mediating its expression by glucocorticoids

The purpose of this study was to determine the pharmacokinetics and absolute bioavailability of cisapride after intravenous (i.v.) and intragastric (i.g.) administration in healthy, adult horses. Five animals received single doses of 0.1 mg/kg, 0.2 mg/kg and 0.4 mg/kg cisapride by the i.g. route in an open, randomized fashion on different occasions separated by a washout period of at least 48 h. Four of these horses were also given a single i.v. dose of 0.1 mg/kg cisapride. Jugular venous blood was collected periodically up to 24 h after dosing. Plasma cisapride concentrations were measured by high-performance liquid chromatography. There was considerable inter individual variability in pharmacokinetic parameters. The mean (SD) values for systemic clearance (CI) and steady-state volume of distribution (Vss) were 494 (43.6) mL/h/kg and 1471 (578) mL/kg, respectively. Although the rate of cisapride absorption was quite rapid, only about half the i.g. dose was absorbed systemically. The average terminal half-life (t1/2) calculated over three i.g. doses was 2.06 h and that for i.v. administration was 2.12 h. The pharmacokinetics of cisapride from 0.1 mg/kg to 0.4 mg/kg were independent of the i.g. dose.     

Regulation of human B19 parvovirus promoter expression by hGABP (E4TF1) transcription factor

The genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113-210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. In Drosophila SL2 cells, hGABPalpha and -beta stimulated P6 promoter activity, and hGABPalpha/hGABPbeta and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1.     

Excitotoxic brain lesion modifies binding to a USF binding site acting as a negative regulatory element in the Protease nexin-1 promoter

This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC.