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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: In a previous study we showed that a region from −182 to +10 bp in the mouse μ-opioid receptor (MOR) promoter exhibited strong promoter activity. To identify protein-DNA interactions in this fragment, gel shift and DNase I footprint analyses were performed using nuclear extracts from mouse brain and the human neuroblastoma cell line, SK-N-SH. Two regions, nucleotide (nt) −121 to −100 and nt −42 to −22, were identified as being specific protein binding sites. The protein-DNA interaction in the nt −42 to −22 region was characterized in detail in this study. Methylation interference analysis of this region showed that nuclear protein from SK-N-SH cells contacted nucleotides within the sequence ATG-CAAAT, which is a binding motif for octamer trans-acting factors. An octamer-1 (Oct-1)-specific antibody supershifted the protein-DNA complex in a gel shift assay. A UV cross-linking experiment showed that a nuclear protein, whose molecular weight is similar to that of the Oct-1 factor, bound to the octamer element in the nt −42 to −22 region. Mutagenesis of four base pairs within the octamer cis-acting element eliminated the specific protein binding in vitro. When the MOR-luciferase reporter construct (−182 to +10 bp) with the same four base pairs mutated was transiently transfected into SK-N-SH cells, a 200% increase in transcriptional activity was observed. Collectively, these data suggest that Oct-1 is binding to the octamer motif in the MOR gene and negatively modulating MOR gene expression.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Apolipoprotein E (apoE) and apoJ are lipid carriers produced in the brain primarily by glial cells. A variety of glial-activating stimuli induce a parallel upregulation of both apolipoproteins expression in vivo and in vitro. To further characterize the cell type and mechanisms by which apoE and apoJ expression are upregulated in activated glia, mixed glial cultures from neonatal rat cortex were treated with the endotoxin lipopolysaccharide (LPS). LPS induced dose-dependent increases in apoJ and decreases in apoE expression and secretion with maximum effects at 1–10 ng/mL and 0.1–1 µg/mL, respectively. Experiments with enriched astroglial and microglial cultures demonstrated that apoE and apoJ expression are predominantly microglial and astroglial, respectively. Given the pivotal role that nuclear factor-κB (NF-κB) plays in glial activation, we assessed its possible role in mediating apoE and apoJ expression by activated glia. LPS robustly increased NF-κB activation in mixed glial cultures. Two NF-κB inhibitors, aspirin (10 mm) and MG-132 (0.1 µm), blocked basal apoE and apoJ secretion as well as LPS-induced apoJ secretion. These data demonstrate that glial apoE and apoJ expression are independently regulated by LPS in microglia and astroglia, respectively, and that activated microglia are the predominant source of apoE in mixed glial cultures. The transcription factor NF-κB appears to be a critical mediator of LPS-stimulated apoJ expression from astroglia.
    Type of Medium: Electronic Resource
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