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  • Epidermal growth factor-mediated inhibition of follicle-stimulating hormone-stimulated StAR gene expression in porcine granulosa cells is associated with reduced histone ... 15590903

    Steroidogenic acute regulatory protein (StAR) mediates cholesterol transport into the mitochondria and is essential for ovarian steroidogenesis. Epidermal growth factor (EGF) has been reported to inhibit FSH-stimulated differentiation in porcine granulosa cells. Previous studies have demonstrated FSH stimulates StAR mRNA accumulation and gene promoter activation in granulosa cells. Treatment of granulosa cells with FSH (5 ng/ml, 6 h) increased StAR mRNA, whereas coaddition of EGF (10 ng/ ml) significantly reduced (P less than 0.05) FSH-stimulated mRNA accumulation by 62.7% +/- 13.9%. Under these same conditions, FSH-stimulated cAMP accumulation in cultures was unaltered by coincubation with EGF. RNA stability studies showed that cotreatment with FSH and EGF did not alter the StAR mRNA half-life compared with FSH alone, t(1/2) = 1.9-3.8 and 2.7-4.1 h, respectively. EGF significantly inhibited (P less than 0.05) FSH-stimulated StAR heterogeneous nuclear RNA levels by 47.6% +/- 6.8 %, implicating a repressive effect on transcription. Surprisingly, EGF (1-50 ng/ml) did not affect FSH stimulation of a 1423-base pair StAR gene promoter-luciferase construct in transient transfection assays in porcine granulosa cells. To evaluate FSH and EGF effects on the endogenous StAR gene, chromatin immunoprecipitation assays were performed in combination with real-time polymerase chain reaction. FSH increased histone H3 acetylation (lysines 9, 14) within the proximal region of the StAR gene promoter and coincubation with EGF blocked this effect. Dimethylation (lysine 9) of histone H3 was not influenced by treatments. In conclusion, EGF repression of FSH-stimulated StAR transcription in porcine granulosa cells is accompanied by reductions in histone H3 acetylation associated with the StAR gene promoter.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-599
    Nombre del producto:
    Anti-acetyl-Histone H3 Antibody

  • Neurofilament proteins and cAMP pathway in brains of mu-, delta- or kappa-opioid receptor gene knock-out mice: effects of chronic morphine administration. 14975676

    Opiate addiction is associated with abnormalities of neurofilament (NF) proteins and upregulation of cAMP signaling in the brain, which may modulate neuronal plasticity. This study investigated, using gene-targeted mice lacking mu-, delta- or kappa-opioid receptors, the role of these receptors in modulating the basal activity and the chronic effects of morphine on both intracellular targets. In WT mice, chronic treatment (5 days) with morphine (20-100 mg/kg) resulted in decreases in the immunodensity of neurofilament (NF)-L in the cerebral cortex (14-23%). In contrast, chronic morphine did not decrease NF-L in cortices of mu-, delta-, and kappa-KO mice, suggesting the involvement of the three types of opioid receptors in this effect of morphine. Also, the marked increase in phosphorylated NF-H induced by chronic morphine in WT mice (two-fold) was abolished in mu -KO mice. In cortex and/or striatum of mu-, delta- and kappa-KO mice, the basal immunodensities of Galphai1/2 proteins, the catalytic isoform (Calpha) of protein kinase A (PKA) and the total content of cAMP response element-binding protein (CREB, the nuclear target of PKA) were not different from those of WT mice. In contrast, phosphorylated CREB (the active form of this transcription factor) was reduced in cortex and/or striatum (23-26%) of mu- and delta-KO mice, but not in kappa-KO animals. These results suggest that the endogenous opioid tone acting on mu-/delta-receptors tonically stimulate CREB activation in the brain. In cortex and/or striatum of WT mice, chronic morphine did not induce upregulation of the main components of the cAMP signaling pathway. In contrast, chronic morphine treatment in mu-KO mice, but not in delta- or kappa-KO, resulted in a paradoxical upregulation of Galphai1/2 (12-19%), PKA (19-21%,) and phosphorylated CREB (21-73%), but not total CREB, in cortex and/or striatum. The induction of heterologous receptor adaptations in mu-KO mice may explain this paradoxical effect of morphine.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-863
    Nombre del producto:
    Anti-CREB Antibody
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