The Journal of biological chemistry
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General anesthetics allosterically modulate the activity of neuronal gamma-aminobutyric acid, type A (GABAA), receptors. Previous mutational studies from our laboratory and others have shown that the regions in transmembrane domain 1 (M1) and pre-M1 of alpha and beta subunits in GABA receptors are essential for positive modulation of GABA binding and function by the intravenous (IV) general anesthetics. Mutation of beta2Gly-219 to Phe corresponded in rho nearly eliminated the modulatory effect of IV anesthetics in alpha1/beta2/gamma2S combination. ⋯ Compared with a 2-3-fold response in wild type, pentobarbital and propofol enhanced less than 0.5-fold; etomidate and alphaxalone modulation was reduced from more than 4- to 1-fold in G219F, G219Y, and G219W. A linear correlation was observed between the volume of the residue at position 219 and the loss of modulation. An identical correlation was found for the effect of modulation on left-shift in the GABA EC50 value; furthermore, the same rank order of residues, related to size, was found for reduction in the maximal direct channel-gating by pentobarbital (1 mm) and etomidate (100 mum) and for increased apparent affinity for direct gating by the IV anesthetics.
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Glycogen synthase kinase-3beta (GSK-3beta) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3beta activity opposes Akt function we used various methods to alleviate GSK-3beta suppression in prostate carcinoma cells. In some experiments, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (a kinase involved in activating Akt) and tumor necrosis factor-alpha (TNF-alpha) were used to activate GSK-3beta. ⋯ When transcription factors activator protein-1 and cyclic AMP-response element (CRE)-binding protein were analyzed as targets of GSK-3beta activity, overexpression of wild-type GSK-3beta suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3betadelta9 caused an (80-fold) increase in CRE-mediated transcription, which was further amplified (up to 130-fold) by combining GSK-3betadelta9 overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3betadelta9 results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei.
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Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. ⋯ These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.