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Electrophoresis Mobility Shift (electrophoresis + mobility_shift)
Selected AbstractsCharacterization of the testis-specific promoter region in the human pituitary adenylate cyclase-activating polypeptide (PACAP) geneGENES TO CELLS, Issue 6 2010Aiko Tominaga Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5,-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP. [source] Identification of a 251-bp Fragment of the PAI-1 Gene Promoter That Mediates the Ethanol-Induced Suppression of PAI-1 ExpressionALCOHOLISM, Issue 5 2001Hernan E. Grenett Background: Moderate alcohol consumption reduces the risk for coronary heart disease. This cardioprotection may be due to ethanol enhancement of fibrinolysis. Fibrinolysis involves the interaction of plasminogen activators (PAs) and the plasminogen activator inhibitor type-1 (PAI-1). Factor(s) that decrease endothelial cell (EC) PAI-1 expression increase fibrinolysis and may decrease the risk for cardiovascular disease. Methods: Five promoter deletion fragments were generated from a 1.1-kb PAI-1 promoter fragment and ligated to a luciferase reporter gene. Cultured human umbilical vein endothelial cells (HUVECs) were transiently transfected with these PAI-1 deletion constructs. A 251-base pair (bp) fragment of the PAI-1 promoter, positions ,800 to ,549, was cloned upstream of a heterologous promoter/enhancer. ECs luciferase activity was measured in the absence/presence of 20 mM ethanol. Electrophoresis mobility shift assays were performed with nuclear extracts from untreated and ethanol-treated ECs using this 251-bp fragment. Results: Deletion analysis showed a region between position ,800 and ,549 mediated ethanol repression of luciferase activity. This 251-bp promoter fragment also repressed the activity of a heterologous promoter/enhancer in the presence of ethanol. Using the labeled 251-bp fragment, nuclear extracts from ethanol-treated ECs contained two inducible bands and one enhanced band. Non-ethanol treated nuclear extracts also contained a band that was not observed in ethanol-treated samples. Competition using 100-fold molar excess of unlabeled probe abolished these four bands. Conclusions: Repression of PAI-I gene transcription in cultured HUVECs exposed to ethanol may involve the interaction of several transcription factors with binding sites localized between positions ,800 and ,549 of the PAI-1 gene promoter. [source] Identification of ERR, as a specific partner of PGC-1, for the activation of PDK4 gene expression in muscleFEBS JOURNAL, Issue 8 2006Makoto Araki Pyruvate dehydrogenase kinase 4 (PDK4) is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions. Transcription of the PDK4 gene is activated by fasting or by the administration of a PPAR, ligand in a tissue-specific manner. Here, we show that the two mechanisms are independent, and that ERR, is directly involved in PPAR,-independent transcriptional activation of the PDK4 gene with PGC-1, as a specific partner. This conclusion is based on the following evidence. First, detailed mutation analyses of the cloned PDK4 gene promoter sequence identified a possible ERR,-binding motif as the PGC-1, responsive element. Second, overexpression of ERR, by cotransfection enhanced, and the knockout of it by shRNAs diminished, PGC-1,-dependent activation. Third, specific binding of ERR, to the identified PGC-1, responsive sequence was confirmed by the electrophoresis mobility shift assay. Finally, cell-type-specific responsiveness to PGC-1, was observed and this could be explained by differences in the expression levels of ERR,, however, ectopic expression of ERR, in poorly responsive cells did not restore PGC-1, responsiveness, indicating that ERR, is necessary, but not sufficient for the response. [source] Functionally important structural elements of the cyanobacterial clock-related protein PexGENES TO CELLS, Issue 1 2009Shunsuke Kurosawa Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the ,3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the ,3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its ,3 helix is probably important to its stability. [source] Atorvastatin induces apoptosis by a caspase-9-dependent pathway: an in vitro study on activated rat hepatic stellate cellsLIVER INTERNATIONAL, Issue 4 2008Isabella Aprigliano Abstract Background: Statins are shown to have cholesterol-independent properties such as anti-inflammation and immunomodulation. Activated hepatic stellate cells (HSCs) acquire the capacity to synthesize matrix proteins in damaged liver. We tested the hypothesis that atorvastatin may be capable of inducing apoptosis in HSCs. Methods: Primary cultures of rat HSCs were exposed to atorvastatin, mevalonic acid and U0126. Quantification of living, apoptotic and necrotic HSCs was performed by flow cytometry and laser-scan microscopy. Cell-cycle analysis was performed by flow cytometry. Pro- and anti-apoptotic factors were investigated by Western blot and electrophoresis mobility shift assay. Protease activity of caspases was calculated using a colorimetric kit. Results: Atorvastatin leads to a G2-arrest and induces apoptosis in activated HSCs. Atorvastatin-mediated apoptosis could be blocked by co-administration of mevalonic acid and U0126. No effects of atorvastatin on gene expression of CD95, CD95L, NF-,B, p53 and p21WAF1 could be observed. Atorvastatin-induced apoptosis in activated HSCs is related to an increased protease activity of caspase-9 and -3. Gene expression of the major proteins of the bcl-system shows that truncated Bid is involved in apoptosis mediated by atorvastatin. By blocking the extracellular signal-regulated protein kinase (ERK1/2) activation by adding U0126, we could prevent the apoptosis induced by atorvastatin. By Western blot we could not detect any change in the activation of c-jun N-terminal kinase (JNK). Conclusions: Atorvastatin induces apoptosis in activated HSCs acting through an ERK-dependent cleavage of Bid and a highly increased protease activity of caspase-9 and -3. JNK is not involved in atorvastatin-mediated apoptosis in HSCs. [source] | ||||||||||