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Gel Shift (gel + shift)
Selected AbstractsCharacterization of the mouse adenylyl cyclase type VIII gene promoter: regulation by cAMP and CREBEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2002Jennifer R. Chao Abstract Adenylyl cyclase (AC) type VIII has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. In the present study, we directly examined the role for the transcription factor CREB (cAMP response element binding protein) in regulating ACVIII expression by cloning a 5.2 kilobase region upstream of the translation start site of the mouse ACVIII gene. Analysis of this fragment revealed consensus elements for several transcription factors, including a canonical cAMP response element (CRE) in close proximity to the transcription initiation region. Next, ACVIII promoter activity was studied in two neural-derived cell lines and in primary cultures of rat striatal neurons. Activation of the cAMP pathway by forskolin treatment increased promoter activity, and a series of deletion and point mutants demonstrated that this activation is mediated specifically via the canonical CRE site. Gel shift assays confirmed that this site can bind CREB and several CREB family proteins. Further, activation of the ACVIII promoter by forskolin was potentiated by expression of a constitutively active form of CREB, CREB-VP16, whereas it was inhibited by expression of a dominant-negative form of CREB, A-CREB. Finally, over-expression of CREB in vivo, by viral-mediated gene transfer, induced ACVIII promoter activity in the brains of ACVIII-LacZ transgenic mice. These results suggest that the ACVIII gene is regulated by CREB in vitro and in vivo and that this regulation may contribute to CREB-dependent neural plasticity. [source] LIN54 is an essential core subunit of the DREAM/LINC complex that binds to the cdc2 promoter in a sequence-specific mannerFEBS JOURNAL, Issue 19 2009Fabienne Schmit Recently, the conserved human LINC/DREAM complex has been described as an important regulator of cell cycle genes. LINC consists of a core module that dynamically associates with E2F transcription factors, p130 and the B-MYB transcription factor in a cell cycle-dependent manner. In this study, we analyzed the evolutionary conserved LIN54 subunit of LINC. We found that LIN54 is required for cell cycle progression. Protein interaction studies demonstrated that a predicted helix,coil,helix motif is required for the interaction of LIN54 with p130 and B-MYB. In addition, we found that the cysteine-rich CXC domain of LIN54 is a novel DNA-binding domain that binds to the cdc2 promoter in a sequence-specific manner. We identified two binding sites for LIN54 in the cdc2 promoter, one of which overlaps with the cell cycle homology region at the transcriptional start site. Gel shift assays suggested that, in quiescent cells, the binding of LIN54 at the cell cycle homology region is stabilized by the binding of E2F4 to the adjacent cell cycle-dependent element. Our data demonstrate that LIN54 is an important and integral subunit of LINC. Structured digital abstract ,,MINT-7239362: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with p130 (uniprotkb:Q08999) by anti tag coimmunoprecipitation (MI:0007) ,,MINT-7239376: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with B-Myb (uniprotkb:P10244) by anti tag coimmunoprecipitation (MI:0007) [source] MIDA1 is a sequence specific DNA binding protein with novel DNA binding propertiesGENES TO CELLS, Issue 9 2000Toshiaki Inoue Background Id proteins not only regulate cell differentiation negatively, but they also promote growth and apoptosis. To know the mechanism of how Id regulates cell fate, we previously isolated an Id-associating protein, MIDA1, which positively regulates cell growth. Its predicted amino acid sequence contains tryptophan-mediated repeats (Tryp-med repeats) similar to the DNA binding region of the c-Myb oncoprotein. We determined whether MIDA1 can bind to DNA in a sequence specific manner by PCR-assisted binding site selection. Results We identified a 7-base sequence (GTCAAGC) surrounded by a 1,3 bp palindromic sequence as the DNA sequence recognized by the Tryp-med repeats of MIDA1. This motif is located within the 5,-flanking sequence of several growth regulating genes. Gel shift assays revealed that this sequence and a certain length of flanking DNA are necessary for MIDA1 to bind DNA in a stable manner. Methylation interference and DNase I footprint analysis suggested that the DNA binding of MIDA1 is resistant to DNA methylation and that MIDA1 does not specifically localize on this particular motif. Conclusions We concluded that MIDA1 is a novel sequence-specific DNA binding protein with some different properties from the usual transcription factors and that MIDA1 may act as a mediator of Id-mediated growth-promoting function through its DNA binding activity. [source] Roles of CmpR, a LysR family transcriptional regulator, in acclimation of the cyanobacterium Synechococcus sp. strain PCC 7942 to low-CO2 and high-light conditionsMOLECULAR MICROBIOLOGY, Issue 3 2004Yukari Takahashi Summary The cmp operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is induced under low CO2 conditions by a LysR family protein CmpR. CmpR was found to be required also for induction of the operon by transfer of the cells from low-light to high-light conditions, indicating involvement of a common mechanism in the high-light- and low-CO2 -responsive regulation. Expression of the high-light inducible genes psbAII and psbAIII, on the other hand, was found to be induced also by low-CO2 conditions. A single promoter was responsible for the high-light and low-CO2 induction of each of psbAII and psbAIII, suggesting involvement of a common regulatory mechanism in the light and CO2 responses of the psbA genes. CmpR was, however, not required for the induction of psbAII and psbAIII, indicating the presence of multiple mechanisms for induction of genes under high-light and low-CO2 conditions. The CmpR-deficient mutant nevertheless showed lower levels of the psbAII and psbAIII transcripts than the wild-type strain under all the light and CO2 conditions examined. Gel shift assays showed that CmpR binds to the enhancer elements of psbAII and psbAIII, through specific interaction with a sequence signature conforming to the binding motif of similar LysR family proteins. These findings showed that CmpR acts as a trans -acting factor that enhances transcription of the photosystem II genes involved in acclimation to high light, revealing a complex network of gene regulation in the cyanobacterium. [source] Novel mode of transcription regulation by SdiA, an Escherichia coli homologue of the quorum-sensing regulatorMOLECULAR MICROBIOLOGY, Issue 5 2001Kaneyoshi Yamamoto SdiA, an Escherichia coli homologue of the quorum-sensing regulator, controls the expression of the ftsQAZ operon for cell division. Transcription of ftsQ is under the control of two promoters, upstream ftsQP2 and downstream ftsQP1, which are separated by 125 bp. SdiA activates transcription from ftsQP2 in vivo. Here, we demonstrate that SdiA facilitates the RNA polymerase binding to ftsQP2 and thereby stimulates transcription from P2. Gel shift and DNase I footprinting assays indicated that SdiA binds to the ftsQP2 promoter region between ,51 and ,25 with respect to the P2 promoter. Activation of ftsQP2 transcription by SdiA was observed with a mutant RNA polymerase containing a C-terminal domain (CTD)-deleted ,-subunit (,235) but not with RNA polymerase containing ,S or a CTD-deleted ,D (,D529). In good agreement with the transcription assay, no protection of P2 was observed with the RNA polymerase holoenzymes, E,S and E,D529. These observations together indicate that: (i) SdiA supports the RNA polymerase binding to ftsQP2; and (ii) this recruitment of RNA polymerase by SdiA depends on the presence of intact ,CTD. This is in contrast to the well-known mechanism of RNA polymerase recruitment by protein,protein contact between class I factors and ,CTD. In addition to the P2 activation, SdiA inhibited RNA polymerase binding to the ftsQP1 promoter and thereby repressed transcription from P1. Gel shift assays indicate weak binding of SdiA to the P1 promoter region downstream from ,13 (or +112 with respect to P2). Neither ,CTD nor ,CTD are required for this inhibition. Thus, the transcription repression of P1 by SdiA may result from its competition with the RNA polymerase in binding to this promoter. [source] Role of the N-terminal Region in the Function of the Photosynthetic Bacterium Transcription Regulator PpsR,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Yoichi Yamazaki PpsR is a transcription repressor for the gene cluster encoding photosystem genes in Rhodobacter sphaeroides. Repression activity is accomplished by DNA binding on the promoter regions of the photosystem gene clusters, and depends on both the redox potential and the presence of antirepressor protein AppA. To understand DNA repression regulation by PpsR, we investigated the function of PpsR domains in self-association for DNA binding. We constructed domain-deletion mutants and verified DNA-binding activity and dimer formation. Gel shift assay for measuring the DNA-binding activity of three sequential N-terminal deletion mutants revealed that N-terminal deletions (of minimum 121 residues) caused loss of binding activity. Size-exclusion gel chromatography revealed that deletion mutant which lacks the N-terminal 121-amino acid deletion mutant to exist as a dimer, although it was less stable than the intact PpsR. The mutants lacking the adjacent regions, Q-linker region and the first Per-Ant-Sim domain, did not form dimers, suggesting the involvement of the N-terminal region in dimer formation. This region is thus considered to be a functional domain in self-association, although not yet identified as a structural domain. Circular dichroism spectrum of the N-terminal region fragment exhibited a ,/, structure. We conclude that this region is a structural and functional domain, contributing to PpsR repression through dimer stabilization. [source] Craniosynostosis-Associated Gene Nell-1 Is Regulated by Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007Thien Truong Abstract We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. Introduction: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5, flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. Materials and Methods: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2,/, calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. Results: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. Conclusions: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis. [source] High glucose activates pituitary proopiomelanocortin gene expression: possible role of free radical-sensitive transcription factorsDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 4 2007Koichi Asaba Abstract Background Hyperglycemia is recognized as a metabolic stress, and indeed it is known to stimulate hypothalamo-pituitary-adrenal (HPA) axis, a representative anti-stress system, in patients with diabetes mellitus or in animal models of hyperglycemia. Thus, we tried to clarify the molecular mechanism of glucose-induced HPA axis activation. Methods We studied the effect of high glucose on the transcriptional regulation of proopiomelanocortin (POMC) gene that encodes adrenocorticotropic hormone, a central mediator of HPA axis, using AtT20 corticotroph cell line in vitro. Results We found that high glucose concentration (24 mM) significantly stimulated the 5,-promoter activity of POMC gene. The effect was promoter-specific, and was mimicked by nuclear factor-kappaB (NF-,B)- or AP1-responsive promoters but not by cAMP-responsive element or serum-response element-containing promoters. Furthermore, the stimulatory effect of high glucose on POMC gene was eliminated by NF-,B and AP1 inhibitors, suggesting the involvement of the transcriptional factors. The POMC 5,-promoter has the canonical NF-,B consensus sequence, and gel shift assay showed the binding of NF-,B to the element. Finally, the effect of high glucose was completely abolished by treatment with a radical quencher 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). Conclusions Our data suggest that hyperglycemia activates POMC gene expression, at least partly, via NF-,B/AP1, and that high-glucose-induced free radical generation may mediate the activation of these transcription factors, which in turn stimulates the transcription of POMC gene. Copyright © 2006 John Wiley & Sons, Ltd. [source] Cadmium blocks hypoxia-inducible factor (HIF)-1-mediated response to hypoxia by stimulating the proteasome-dependent degradation of HIF-1,FEBS JOURNAL, Issue 13 2000Yang-Sook Chun Cadmium is a substantial industrial and environmental pollutant which seriously impairs erythropoiesis. Cd has been demonstrated to aggravate anemia by suppressing erythropoietin gene expression in anemic patients. As hypoxic induction of erythropoietin mRNA depends on a transcription factor, hypoxia-inducible factor 1 (HIF-1), we hypothesized that Cd suppresses the hypoxic activation of HIF-1. In hypoxic Hep3B cells, all mRNAs of various genes, which are known to be upregulated by HIF-1 activation under hypoxia, were suppressed by Cd in a dose-dependent manner. Cd inhibited the hypoxia-induced activity of luciferase in 293 cells which was transfected with a reporter plasmid carrying a hypoxia response element. By electrophoretic mobility gel shift assay, Cd inhibited the DNA-binding activity of HIF-1 in hypoxic Hep3B cells. Cd reduced the amount of HIF-1, protein in hypoxia, whereas it didn't affect HIF-1 , mRNA levels. Moreover, Cd inhibited HIF-1, accumulation induced by cobalt and desferrioxamine. Antioxidants and a proteasome inhibitor prevented the HIF-1, degradation caused by Cd. The possibility that oxidative stress mediates this action of Cd was examined. Cd didn't affect protein oxidation and reduced glutathione levels in hypoxic cells. These results indicate that Cd triggers a redox/proteasome-dependent degradation of HIF-1, protein, reducing HIF-1 activity and in turn suppressing the hypoxic induction of hypoxia-inducible genes. [source] Mucosal NOD2 expression and NF-,B activation in pediatric Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 3 2008Laura Stronati PhD Abstract Background: Recent advances in the pathogenesis of Crohn's disease (CD) have suggested that an aberrant innate immune response initiates the cascade of events leading to T-cell activation and to disease development. NOD2 protein, which is mainly expressed by innate immunity cells, appears to play a key role against bacteria by triggering a host defense response through the activation of the transcriptor factor NF-,B and a consequent proinflammatory cytokine production. The present study was aimed at investigating the expression and activity of NOD2, NF-,B, and of 2 proinflammatory cytokines, TNF, and IL-1,, in mucosal biopsies of CD affected children compared to healthy controls. Methods: In all, 22 children with active CD and 10 matched controls were entered in the study. mRNA and protein expressions were detected using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot; NF-,B binding activity was assessed by electromobility gel shift assay (EMSA). Results: NOD2 and IL-1, mRNAs were upregulated in CD children. Protein levels of NOD2, TNF,, and nuclear NF-,B, as well as the binding activity of NF-,B to a consensus DNA sequence, were significantly increased in inflamed mucosa of patients as compared to controls. Moreover, NF-,B activity was strongly upregulated in patients also when bound to the NOD2 promoter site. No difference was seen between patients and controls when NF-,B binding activity was determined in the uninflamed tissue. Conclusions: This study suggests that altered mechanisms regulating NOD2 induction, NF-,B activation and cytokine production may contribute to dysregulate the innate immune response underlying pediatric CD. (Inflamm Bowel Dis 2007) [source] Environmental Toxicants May Modulate Osteoblast Differentiation by a Mechanism Involving the Aryl Hydrocarbon Receptor,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2007Elizabeth P Ryan Abstract The AHR mediates many of the toxicological effects of aromatic hydrocarbons. We show that AHR expression in osteoblasts parallels the induction of early bone-specific genes involved in maturation. The AHR may not only mediate the effects of toxicants, but with an as yet unidentified ligand, be involved in the differentiation pathways of osteoblasts. Introduction: Metabolic bone diseases arise as a result of an imbalance in bone cell activities. Recent evidence suggests that environmental toxicants may be contributing factors altering these activities. One candidate molecule implicated in mediating the toxic effects of exogenous compounds is the aryl hydrocarbon receptor (AHR). Materials and Methods: Osteoblasts isolated from neonatal rat calvaria were analyzed for AHR expression by quantitative PCR, Western blot, and immunohistochemistry. In addition, AHR activation was evaluated by electromobility gel shift assay and fluorescence microscopy. Results: Our findings showed AHR expression in mature osteoblasts in vivo. The pattern of AHR expression peaks after alkaline phosphatase and before induction of osteocalcin. We first show that AHR functions as a transactivating receptor in osteoblasts, as evidenced by its ligand-dependent migration to the nucleus and its association with known dioxin response elements. AHR activation by 2,3,7,8-tetrachlorodibenzo -p -dioxin (TCDD) mediated the induction of cytochrome p450 1A1 and cycloxygenase-2 protein levels. This effect could be inhibited by the potent AHR antagonist, 3,4 methoxynitroflavone. Furthermore, lead treatment of osteoblasts upregulates the expression of AHR mRNA and protein levels, supporting a novel mechanism whereby lead in the skeleton may increase the sensitivity of bone cells to toxicant exposure. Conclusions: These data imply that the AHR mediates the effects of aromatic toxicants on bone and that AHR expression is regulated during osteoblast differentiation. [source] MEK/ERK Signaling Controls Osmoregulation of Nucleus Pulposus Cells of the Intervertebral Disc by Transactivation of TonEBP/OREBP,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2007Tsung-Ting Tsai Abstract Earlier studies have shown that intervertebral disc cells express TonEBP, a transcription factor that permits adaptation to osmotic stress and regulates aggrecan gene expression. However, the mechanism of hyperosmotic activation of TonEBP in disc cells is not known. Results of this study show that hypertonic activation of ERK signaling regulates transactivation activity of TonEBP, modulating its function. Introduction: In an earlier report, we showed that tonicity enhancer binding protein (TonEBP) positively regulates aggrecan gene expression in disc cells, thereby autoregulating its osmotic environment. Although these studies indicated that the cells of the nucleus pulposus were optimally adapted to a hyperosmotic state, the mechanism by which the cells transduce the osmotic stress was not delineated. The primary goal of this study was to test the hypothesis that, in a hyperosmotic medium, the extracellular signal-regulated kinase (ERK) signaling pathway regulated TonEBP activity. Materials and Methods: Nucleus pulposus cells were maintained in isotonic or hypertonic media, and MAPK activation and TonEBP expression were analyzed. To study the role of MAPK in regulation of TonEBP function, gel shift and luciferase reporter assays were performed. ERK expression in cells was modulated by using expression plasmids or siRNA, and transactivation domain (TAD)-TonEBP activity was studied. Results: We found that hypertonicity resulted in phosphorylation and activation of ERK1/2 proteins and concomitant activation of C terminus TAD activity of ELK-1, a downstream transcription factor. In hypertonic media, treatment with ERK and p38 inhibitors resulted in downregulation of TonE promoter activity of TauT and HSP-70 and decreased binding of TonEBP to TonE motif. Similarly, forced expression of DN-ERK and DN-p38 in nucleus pulposus cells suppressed TauT and HSP-70 reporter gene activity. Finally, we noted that ERK was needed for transactivation of TonEBP. Expression of DN-ERK significantly suppressed, whereas, WT-ERK and CA-MEK1 enhanced, TAD activity of TonEBP. Experiments performed with HeLa cells indicated that the ERK signaling pathway also served a major role in regulating the osmotic response in nondiscal cells. Conclusions: Together, these studies showed that adaptation of the nucleus pulposus cells to their hyperosmotic milieu is dependent on activation of the ERK and p38- MAPK pathways acting through TonEBP and its target genes. [source] Tristetraprolin recruits functional mRNA decay complexes to ARE sequencesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2007Heidi H. Hau Abstract AU-rich elements (AREs) in the 3, untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE-binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleotide ARE sequences from the TNF-alpha 3, UTR promoted TTP-dependent mRNA decay when it was inserted into the 3, UTR of a beta-globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP-containing complex in cytoplasmic extracts from TTP-transfected HeLa cells that bound specifically to short ARE sequences. This TTP-containing complex also contained the 5,,3, exonuclease Xrn1 and the exosome component PM-scl75 because it was super-shifted with anti-Xrn1 or anti-PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP-dependent manner. Using a competition binding assay, we found that the TTP-containing complex bound with high affinity to short ARE sequences from GM-CSF, IL-3, TNF-alpha, IL-2, and c-fos, but did not bind to a U-rich sequence from c-myc, a 22 nucleotide poly U sequence or a mutated GM-CSF control sequence. High affinity binding by the TTP-containing complex correlated with TTP-dependent deadenylation and decay of capped, polyadenylated transcripts in a cell-free mRNA decay assay, suggesting that the TTP-containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript. J. Cell. Biochem. 100: 1477,1492, 2007. © 2006 Wiley-Liss, Inc. [source] Salvianolic acid B attenuates plasminogen activator inhibitor type 1 production in TNF-, treated human umbilical vein endothelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Zhe Zhou Abstract Plasminogen activator inhibitor type 1 (PAI-1), which plays a role in the development of atherosclerosis, is produced by endothelial cells following stimulation with various inflammatory cytokines such as tumor necrosis factor (TNF-,). In the present study, we investigated the effects of a potent water-soluble antioxidant, salvianolic acid B (SalB; derived from the Chinese herb, Salviamiltiorrhiza), on the expression of PAI-1 in TNF-,-treated human umbilical vein endothelial cells (HUVECs). We found that SalB inhibited TNF-,-induced PAI-1 mRNA production and protein secretion in HUVECs. Treatment with SalB (0.05 and 0.15 µM) notably attenuated TNF-, induced expression of PAI-1 to 90.5% and 74.6%, respectively, after 12 h, and to 75.1% and 64.2%, respectively, after 18 h. We also observed a dose-dependent decrease in PAI-1 protein production in the presence of SalB. We then used pathway inhibitors to investigate which step of the TNF-, induced signaling pathway was targeted by SalB. We found that the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, increased the inhibitory effects of SalB on TNF-,-induced PAI-1 secretion, whereas the nuclear factor-,B (NF-,B) inhibitor, emodin, and the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, did not. A gel shift assay further showed that SalB inhibited the TNF-,-activated NF-,B and AP-1 DNA binding activities in a dose-dependent manner. Collectively, these results indicate that the NF-,B and ERK-AP-1 pathways are possible targets of SalB in the regulation of TNF-,-stimulated PAI-1 production in HUVECs. © 2005 Wiley-Liss, Inc. [source] Ets protein Elf-1 bidirectionally suppresses transcriptional activities of the tumor suppressor Tsc2 gene and the repair-related Nth1 geneMOLECULAR CARCINOGENESIS, Issue 3 2003Satoshi Honda Abstract Alterations in the rat tuberous sclerosis gene (Tsc2) cause renal cell carcinomas (RCCs) with complete penetrance. In this study, it was shown that the minimal core promoters of the rat Tsc2 and endonuclease III 1 (Nth1) genes, lying in a 5,-to-5, arrangement, were localized in a 0.11-kb region containing two Ets binding sites (EBSs). This region worked as a bidirectional promoter in a single reporter plasmid. Mutational inactivation of each of the two EBSs significantly reduced promoter activity. Moreover, gel shift assays revealed the presence of specific EBSs-protein complexes. These results demonstrate that some members of the Ets family positively regulate the promoter activities of the Tsc2/Nth1 genes by binding to the EBSs. We identified Elf-1 as a binding factor for EBSs through super-shift assays, and detected ,35 kDa bands with an EBSs-containing DNA probe by Southwestern blot analysis. Forced expression of Elf-1 in cells, however, bidirectionally suppressed the activities of the Tsc2/Nth1 promoters. Elf-1 may be a negative regulator of Tsc2/Nth1 gene expression and may compete against positive regulators for binding to the EBSs. Our observations suggest that mechanisms that inactivate Tsc2 gene expression, such as promoter suppression, may exist. © 2003 Wiley-Liss, Inc. [source] Mechanism of low CO2 -induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942MOLECULAR MICROBIOLOGY, Issue 1 2008Takashi Nishimura Summary The cmp operon of the cyanobacterium Synechococcus elongatus strain PCC 7942, encoding the subunits of the ABC-type bicarbonate transporter, is activated under CO2 -limited growth conditions in a manner dependent on CmpR, a LysR family transcription factor of CbbR subfamily. The 0.7 kb long regulatory region of the operon carried a single promoter, which responded to CO2 limitation. Using the luxAB reporter system, three cis -acting elements involved in the low-CO2 activation of transcription, each consisting of a pair of LysR recognition signatures overlapping at their ends, were identified in the regulatory region. CmpR was shown to bind to the regulatory region, yielding several DNA,protein complexes in gel shift assays. Addition of ribulose-1,5-bisphosphate (> 1 mM) or 2-phosphoglycolate (> 10 ,M) enhanced the binding of CmpR in a concentration-dependent manner, promoting formation of large DNA,protein complexes. Given the involvement of O2 in adaptive responses of cyanobacteria to low-CO2 conditions, our results suggest that 2-phosphoglycolate, which is produced by oxygenation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of ribulose-1,5-bisphosphate under CO2 -limited conditions, acts as the co-inducer in the activation of the cmp operon by CmpR. [source] A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gelsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2003Debra Jing Abstract DNA-binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein-nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous 32P-labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility-shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR® Green EMSA dye, while proteins are subsequently detected with SYPRO® Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein-nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid. [source] The DDF1 transcriptional activator upregulates expression of a gibberellin-deactivating gene, GA2ox7, under high-salinity stress in ArabidopsisTHE PLANT JOURNAL, Issue 4 2008Hiroshi Magome Summary High-salinity stress affects plant growth and development. We have previously reported that overexpression of the salinity-responsive DWARF AND DELAYED FLOWERING 1 (DDF1) gene, encoding an AP2 transcription factor of the DREB1/CBF subfamily, causes dwarfism mainly by levels of reducing bioactive gibberellin (GA) in transgenic Arabidopsis. Here, we found that the GA 2-oxidase 7 gene (GA2ox7), which encodes a C20 -GA deactivation enzyme, is strongly upregulated in DDF1 -overexpressing transgenic plants. A loss-of-function mutation of GA2ox7 (ga2ox7-2) suppressed the dwarf phenotype of DDF1 -overexpressing plants, indicating that their GA deficiency is due to overexpression of GA2ox7. Transient overexpression of DDF1 activated the promoter of GA2ox7 in Arabidopsis leaves. A gel shift assay showed that DDF1 binds DRE-like motifs (GCCGAC and ATCGAC) in the GA2ox7 promoter. In Arabidopsis under high-salinity stress, six GA2ox genes, including GA2ox7, were upregulated. Furthermore, the ga2ox7-2 mutant was less growth retarded than wild-type Col under high-salinity stress. These results demonstrate that, under salinity stress, Arabidopsis plants actively reduce endogenous GA levels via the induction of GA 2-oxidase, with the result that growth is repressed for stress adaptation. [source] Ginsenoside Rg3 inhibits phenylephrine-induced vascular contraction through induction of nitric oxide synthaseBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2003Nak Doo Kim Ginsenoside Rg3 (Rg3) isolated from Panax ginseng relaxes vessels and exerts a cytoprotective effect. In view of the fact that nitric oxide (NO) is involved in vascular hyporeactivity and immunostimulation, the effects of total ginsenosides (GS) and Rg3 on the vascular responses and the expression of inducible nitric oxide synthase (iNOS) were investigated. Vasocontraction of endothelium-denuded aortic ring was induced by phenylephrine with or without GS or Rg3. The expression of iNOS was assessed by Western blot and RT,PCR analyses. NF- ,B activation was monitored by gel shift, immunoblot and immunocytochemical analyses. Incubation of the endothelium-denuded aortic ring with GS or Rg3 inhibited phenylephrine-induced vasocontraction, which was abrogated by NOS inhibition. GS or Rg3 increased NO production in aortic rings, but Rb1, Rc, Re and Rg1 had no effect. Aortic rings obtained from rats treated with GS or Rg3 responded to phenylnephrine to a lesser extent, while producing NO to a larger extent, than those from control animals. GS or Rg3 induced iNOS in vascular smooth muscle. Rg3 induced iNOS with increase in NO production in Raw264.7 cells. Rg3 increased NF- ,B DNA binding, whose band was supershifted with anti-p65 and anti-p50 antibodies, and elicited p65 nuclear translocation, which was accompanied by phosphorylation and degradation of I- ,B,. PKC regulated iNOS induction by Rg3. In conclusion, Rg3 relaxes vessels as a consequence of NO production, to which iNOS induction contributes, and iNOS induction by Rg3 accompanied NF- ,B activation, which involves phosphorylation and degradation of I- ,B, and nuclear translocation of p65. British Journal of Pharmacology (2003) 140, 661,670. doi:10.1038/sj.bjp.0705490 [source] | ||||||||||||||||