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DNA-binding Activity (DNA-bind + activity)

Distribution by Scientific Domains
Life Sciences46%
Medical Sciences36%
Chemistry17%

Kinds of DNA-binding Activity

  • b DNA-bind activity
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    Selected Abstracts

    Papillary and muscle invasive bladder tumors with distinct genomic stability profiles have different DNA repair fidelity and KU DNA-binding activities

    GENES, CHROMOSOMES AND CANCER, Issue 4 2009
    Johanne Bentley
    Low-grade noninvasive papillary bladder tumors are genetically stable whereas muscle invasive bladder tumors display high levels of chromosomal aberrations. As cells deficient for nonhomologous end-joining (NHEJ) pathway components display increased genomic instability, we sought to determine the NHEJ repair characteristics of bladder tumors and correlate this with tumor stage and grade. A panel of 13 human bladder tumors of defined stage and grade were investigated for chromosomal aberrations by comparative genomic hybridization and for NHEJ repair fidelity and function. Repair assays were conducted with extracts made directly from bladder tumor specimens to avoid culture-induced phenotypic alterations and selection bias as only a minority of bladder tumors grow in culture. Four noninvasive bladder tumors (pTaG2), which were genetically stable, repaired a partially incompatible double-strand break (DSB) by NHEJ-dependent annealing of termini and fill-in of overhangs with minimal loss of nucleotides. In contrast, four muscle invasive bladder cancers (pT2-3G3), which displayed gross chromosomal rearrangements, repaired DSBs in an error-prone manner involving extensive resection and microhomology association. Four minimally invasive bladder cancers (pT1G3) had characteristics of both repair types. Error-prone repair in bladder tumors correlated with reduced KU DNA-binding and loss of TP53 function. In conclusion, there were distinct differences in DSB repair between noninvasive papillary tumors and higher stage/grade invasive cancers. End-joining fidelity correlated with stage and was increasingly error-prone as tumors became more invasive and KU binding activity reduced; these changes may underlie the different genomic profiles of these tumors. © 2008 Wiley-Liss, Inc. [source]

    Ethanol Impairs Activation of Retinoic Acid Receptors in Cerebellar Granule Cells in a Rodent Model of Fetal Alcohol Spectrum Disorders

    ALCOHOLISM, Issue 5 2010
    Ambrish Kumar
    Background:, Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to ethanol toxic effects. Retinoic acid (RA) is a physiologically active metabolite of vitamin A that is locally synthesized in the cerebellum. Studies have shown that RA is required for neuronal development, but it remains unknown if ethanol impairs RA signaling and thus induces neuronal malformations. In this study, we tested the hypothesis that ethanol impairs the expression and activation of RA receptors in cerebellum and in cerebellar granule cells. Methods:, The cerebellum of ethanol unexposed and exposed pups was used to study the expression of retinoic acid receptors (RARs or RXRs) by immunohistochemistry and by Western blot analysis. We also studied the effect of ethanol on expression of RA receptors in the cerebellar granule cells. Activation of RA receptors (DNA-binding activities) in response to high-dose ethanol was determined by electrophoretic mobility shift and supershift assays. Results:, Findings from these studies demonstrated that ethanol exposure reduced the expression of RAR,/, while it increased the expression of RXR,/, in the cerebellum and in cerebellar granule neurons. Immuno-histological studies further strengthened the expression pattern of RA receptors in response to ethanol. The DNA-binding activity of RARs was reduced, while DNA-binding activity of RXRs was increased in response to ethanol exposure. Conclusion:, For the first time, our studies have demonstrated that high-dose ethanol affects the expression and activation of RA receptors, which could impair the signaling events and induce harmful effects on the survival and differentiation of cerebellar granule cells. Taken together, these findings could provide insight into the treatment options for brain defects caused by excessive ethanol exposure, such as in Fetal Alcohol Spectrum Disorders. [source]

    The DNA-remodelling activity of DnaD is the sum of oligomerization and DNA-binding activities on separate domains

    MOLECULAR MICROBIOLOGY, Issue 4 2006
    Maria J. V. M. Carneiro
    Summary The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C-terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains. [source]

    Cell-free production of transducible transcription factors for nuclear reprogramming,

    BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009
    William C. Yang
    Abstract Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli -based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target. Biotechnol. Bioeng. 2009; 104: 1047,1058. © 2009 Wiley Periodicals, Inc. [source]

    The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR

    ENVIRONMENTAL MICROBIOLOGY, Issue 10 2007
    Tom Defoirdt
    Summary This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxRVh mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxRVh response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxRVh protein also showed less shifts with furanone-treated LuxRVh, whereas the LuxRVh concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxRVh unable to bind to the promoter sequences of quorum sensing-regulated genes. [source]

    Signaling events leading to the curative effect of cystatin on experimental visceral leishmaniasis: Involvement of ERK1/2, NF-,B and JAK/STAT pathways

    EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009
    Susanta Kar
    Abstract Curative effect of cystatin, a natural cystein protease inhibitor, on experimental visceral leishmaniasis was associated with strong upregulation of iNOS. The transductional mechanisms underlying this cellular response was investigated in the murine macrophage cell line RAW 264.7 and in the BALB/c mouse model of visceral leishmaniasis. Cystatin synergizes with IFN-, in inducing ERK1/2 phosphorylation and NF-,B DNA-binding activity. Pretreatment of cells with specific inhibitors of NF-,B or ERK1/2 pathway blocked the cystatin plus IFN-,-inducible NF-,B activity and markedly reduced the expression of iNOS at both mRNA and protein levels. Silencing of mitogen- and stress-activated protein kinase 1 significantly reduced cystatin-mediated NF-,B-dependent iNOS gene transcription suggesting the involvement of mitogen- and stress-activated protein kinase 1 activation in ERK1/2 signaling. DNA binding as well as silencing experiments revealed the requirement of IFN-,-mediated JAK-STAT activation even though cystatin did not modulate this signaling cascade by itself. In the in vivo situation, key steps in the activation cascade of NF-,B, including nuclear translocation of NF-,B subunits, I,B phosphorylation and I,B kinase, are all remarkably enhanced in Leishmania -infected mice by cystatin. Understanding the molecular mechanisms through which cystatin modulates macrophage effector responses will contribute to better define its potential for macrophage-associated diseases, in general. [source]

    Analysis of DNA-binding sites on Mhr1, a yeast mitochondrial ATP-independent homologous pairing protein

    FEBS JOURNAL, Issue 6 2010
    Tokiha Masuda
    The Mhr1 protein is necessary for mtDNA homologous recombination in Saccharomyces cerevisiae. Homologous pairing (HP) is an essential reaction during homologous recombination, and is generally catalyzed by the RecA/Rad51 family of proteins in an ATP-dependent manner. Mhr1 catalyzes HP through a mechanism similar, at the DNA level, to that of the RecA/Rad51 proteins, but without utilizing ATP. However, it has no sequence homology with the RecA/Rad51 family proteins or with other ATP-independent HP proteins, and exhibits different requirements for DNA topology. We are interested in the structural features of the functional domains of Mhr1. In this study, we employed the native fluorescence of Mhr1's Trp residues to examine the energy transfer from the Trp residues to etheno-modified ssDNA bound to Mhr1. Our results showed that two of the seven Trp residues (Trp71 and Trp165) are spatially close to the bound DNA. A systematic analysis of mutant Mhr1 proteins revealed that Asp69 is involved in Mg2+ -dependent DNA binding, and that multiple Lys and Arg residues located around Trp71 and Trp165 are involved in the DNA-binding activity of Mhr1. In addition, in vivo complementation analyses showed that a region around Trp165 is important for the maintenance of mtDNA. On the basis of these results, we discuss the function of the region surrounding Trp165. [source]

    Nuclear import and DNA-binding activity of RFX1

    FEBS JOURNAL, Issue 10 2001
    Evidence for an autoinhibitory mechanism
    RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent ,acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC. [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 2000
    Yang-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]

    Central forkhead domain of human TFIIE, plays a primary role in binding double-stranded DNA at transcription initiation

    GENES TO CELLS, Issue 3 2009
    Aki Tanaka
    The human general transcription factor, TFIIE, consists of two subunits, , and ,. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIE,. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIE, binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function. [source]

    Functionally important structural elements of the cyanobacterial clock-related protein Pex

    GENES TO CELLS, Issue 1 2009
    Shunsuke 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 prevents carbohydrate response element binding protein activation in the fructose-fed rat by activating protein kinase A,

    HEPATOLOGY, Issue 1 2009
    Ricardo Rodríguez-Calvo
    High fructose intake contributes to the overall epidemic of obesity and metabolic disease. Here we examined whether atorvastatin treatment blocks the activation of the carbohydrate response element binding protein (ChREBP) in the fructose-fed rat. Fructose feeding increased blood pressure (21%, P < 0.05), plasma free fatty acids (59%, P < 0.01), and plasma triglyceride levels (129%, P < 0.001) compared with control rats fed standard chow. These increases were prevented by atorvastatin. Rats fed the fructose-rich diet showed enhanced hepatic messenger RNA (mRNA) levels of glycerol-3-phosphate acyltransferase (Gpat1) (1.45-fold induction, P < 0.05), which is the rate-limiting enzyme for the synthesis of triglycerides, and liver triglyceride content (2.35-fold induction, P < 0.001). Drug treatment inhibited the induction of Gpat1 and increased the expression of liver-type carnitine palmitoyltransferase 1 (L-Cpt-1) (128%, P < 0.01). These observations indicate that atorvastatin diverts fatty acids from triglyceride synthesis to fatty acid oxidation, which is consistent with the reduction in liver triglyceride levels (28%, P < 0.01) observed after atorvastatin treatment. The expression of Gpat1 is regulated by ChREBP and sterol regulatory element binding protein-1c (SREBP-1c). Atorvastatin treatment prevented fructose-induced ChREBP translocation and the increase in ChREBP DNA-binding activity while reducing SREBP-1c DNA-binding activity. Statin treatment increased phospho-protein kinase A (PKA), which promotes nuclear exclusion of ChREBP and reduces its DNA-binding activity. Human HepG2 cells exposed to fructose showed enhanced ChREBP DNA-binding activity, which was not observed in the presence of atorvastatin. Furthermore, atorvastatin treatment increased the CPT-I mRNA levels in these cells. Interestingly, both effects of this drug were abolished in the presence of the PKA inhibitor H89. Conclusion: These findings indicate that atorvastatin inhibits fructose-induced ChREBP activity and increases CPT-I expression by activating PKA. (HEPATOLOGY > 2009;49:106-115.) [source]

    Lymphoid enhancer factor interacts with GATA-3 and controls its function in T helper type 2 cells

    IMMUNOLOGY, Issue 3 2008
    Mohammad B. Hossain
    Summary GATA-3 is the master transcription factor for T helper 2 (Th2) cell differentiation and is critical for the expression of Th2 cytokines. Little is known, however, about the nature of the functional molecular complexes of GATA-3. We identified a high-mobility group (HMG)-box type transcription factor, lymphoid enhancer factor 1 (LEF-1), in the GATA-3 complex present in Th2 cells using a Flag-calmodulin-binding peptide (CBP)-tag based proteomics method. The interaction between GATA-3 and LEF-1 was confirmed by co-immunoprecipitation experiments using LEF-1-introduced T-cell lineage TG40 cells. The HMG-box domain of LEF-1 and two zinc finger domains of GATA-3 were found to be important for the physical association. The introduction of LEF-1 into developing Th2 cells resulted in the suppression of Th2 cytokine production. The suppression was significantly lower in the cells into which a HMG-box-deleted LEF-1 mutant was introduced. Moreover, LEF-1 inhibited the binding activity of GATA-3 to the interleukin (IL)-5 promoter. These results suggest that LEF-1 is involved in the GATA-3 complex, while also regulating the GATA-3 function, such as the induction of Th2 cytokine expression via the inhibition of the DNA-binding activity of GATA-3. [source]

    Caffeic acid phenethyl ester decreases cholangiocarcinoma growth by inhibition of NF-,B and induction of apoptosis

    INTERNATIONAL JOURNAL OF CANCER, Issue 3 2009
    Paolo Onori
    Abstract Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF-,B), which is constitutively active in cholangiocarcinoma (CCH) cells. We evaluated the effects of CAPE on CCH growth both in vitro and in vivo. Inhibition of NF-,B DNA-binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 ,M. CAPE decreases the expression of NF-,B1 (p50) and RelA (p65). CAPE decreased the growth of a number of CCH cells but not normal cholangiocytes. Cell cycle decrease was seen by a decrease in PCNA protein expression and the number of BrdU-positive cells treated with CAPE at 20 ,M compared to vehicle. Inhibition of growth and increased cell cycle arrest of Mz-ChA-1 cells by CAPE were coupled with increased apoptosis. Bax expression was increased, whereas Bcl-2 was decreased in cells treated with CAPE compared to vehicle. In vivo studies were performed in BALB/c nude (nu/nu) mice implanted subcutaneously with Mz-ChA-1 cells and treated with daily IP injections of DMSO or CAPE (10 mg/kg body weight in DMSO) for 77 days. Tumor growth was decreased and tumor latency was increased 2-fold in CAPE compared to vehicle-treated nude mice. In tumor samples, decreased CCH growth by CAPE was coupled with increased apoptosis. CAPE both in vivo and in vitro decreases the growth of CCH cells by increasing apoptosis. These results demonstrate that CAPE might be an important therapeutic tool in the treatment of CCH. © 2009 UICC [source]

    Sp1 and Smad3 are required for high glucose-induced p21WAF1 gene transcription in LLC-PK1 cells

    JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2007
    Tsai-Der Chuang
    Abstract The cyclin-dependent kinase inhibitor p21WAF1 is required for diabetic glomerular hypertrophy. High glucose-induced hypertrophy in proximal tubule cells is dependent on transforming growth factor-, (TGF-,). Many of the TGF-,-induced effects are dependent on Smad2/3. Thus, the molecular mechanisms of high glucose-induced p21WAF1 and hypertrophy were studied in high glucose-cultured proximal tubule-like LLC-PK1 cells. We found that high glucose (30 mM) induced hypertrophy at 72 h. High glucose also increased the expression of p21WAF1 protein and p21WAF1 mRNA transcription and abundance at 48 h. The DNA element in the 5, regulatory region of p21WAF1 gene essential for high glucose-induced p21WAF1 gene transcription was identified as Sp1 by a series of the 5, regulatory region of p21WAF1 gene deletion mutants. Moreover, high glucose activated Smad2/3 while increasing the Sp1 DNA-binding activity. High glucose also increased the Sp1-dependent transcriptional activity of p21WAF1 gene. High glucose-induced hypertrophy was attenuated by p21WAF1 short interfering RNA and Smad3 dominant-negative plasmid transfection. We concluded that high glucose induced hypertrophy via Sp1-Smad2/3-dependent activation of p21WAF1 gene transcription in LLC-PK1 cells. J. Cell. Biochem. 102: 1190,1201, 2007. © 2007 Wiley-Liss, Inc. [source]

    Inhibition of CCAAT/enhancer binding protein , expression by chrysin in microglial cells results in anti-inflammatory and neuroprotective effects

    JOURNAL OF NEUROCHEMISTRY, Issue 2 2010
    Núria Gresa-Arribas
    J. Neurochem. (2010) 115, 526,536. Abstract The control of neuroinflammation is a potential target to be considered in the treatment of neurodegenerative diseases. It is therefore important to find anti-inflammatory drugs and study new targets that inhibit neuroinflammation. We designed an experimental model of neuroinflammation in vitro to study the anti-inflammatory and neuroprotective effects of the flavonoid chrysin and the involvement of nuclear factor-,B p65 and CCAAT/enhancer binding proteins (C/EBPs) , and , transcription factors in its mechanism of action. We used primary cultures of mouse embryonic cortical neurons and cultures of BV2 (murine microglial cell line) or mouse primary microglia. We induced neuronal death in neuronal-BV2/microglial co-cultures using lipopolysaccharide of Escherichia coli and interferon-,. Chrysin pre-treatment inhibited nitric oxide and tumor necrosis factor-, production, as well as inducible nitric oxide synthase expression in lipopolysaccharide E. coli and interferon-,-treated microglial cells, but did not affect cyclooxygenase-2 expression. Chrysin pre-treatment also protected neurons against the neurotoxicity induced by reactive microglial cells. These effects were associated to a decrease in C/EBP, protein level, mRNA expression, and DNA-binding activity, with no effect on C/EBP, and p65 nuclear protein levels or DNA-binding activity, pointing out C/EBP, as a possible mediator of chrysin effects. Consequently, C/EBP, is a possible target to act against neuroinflammation in neurodegenerative processes. [source]

    Triptolide inhibits COX-2 expression and PGE2 release by suppressing the activity of NF-,B and JNK in LPS-treated microglia

    JOURNAL OF NEUROCHEMISTRY, Issue 3 2008
    Yuntao Gong
    Abstract Activated microglia participate in neuroinflammation which contributes to neuronal damage in neurodegenerative diseases. Inhibition of microglial activation may have potential anti-inflammatory effects. Our laboratory has previously reported that triptolide, a natural biologically active compound extracted from Tripterygium wilfordii, could protect dopaminergic neurons from inflammation-mediated damage. However, the mechanism by which triptolide inhibits inflammation remains unknown. We reported here that inhibition of prostaglandin E2 (PGE2) production could be a potential mechanism of triptolide to suppress inflammation. Triptolide suppressed c- jun NH2-terminal kinase (JNK) phosphorylation, cyclooxygenase 2 (COX-2) expression and PGE2 production in microglial cultures treated with lipopolysaccharide (LPS). Triptolide also greatly inhibited the transcriptional activity, but not the DNA-binding activity of nuclear factor-,B (NF-,B) in microglia following LPS stimulation. These results indicate that triptolide might suppress NF-,B activity to down-regulate COX-2 expression. The LPS-stimulated transcriptional activity of NF-,B was suppressed by inhibition of p38MAPK, but not by that of JNK and extracellular signal-regulated kinase. Furthermore, the LPS-induced PGE2 production was reduced by inhibiting these kinases. Taken together, these results suggest that triptolide may suppress neuroinflammation via a mechanism that involves inactivation of two parallel signaling pathways: p38-NF-,B-COX-2-PGE2 and JNK-PGE2. [source]

    Involvement of nuclear factor-kappa B in bcl-xL-induced interleukin 8 expression in glioblastoma

    JOURNAL OF NEUROCHEMISTRY, Issue 3 2008
    Chiara Gabellini
    Abstract We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkB, and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkB, protein, paralleled by an increase in the phosphorylation of the same IkB, and IKK,/,. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkB, in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells. [source]

    Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene

    JOURNAL OF NEUROCHEMISTRY, Issue 5 2008
    Anna Dobretsova
    Abstract The myelin proteolipid protein gene (Plp1) encodes the most abundant protein found in CNS myelin, accounting for nearly one-half of the total protein. Its expression in oligodendrocytes is developmentally regulated , peaking during the active myelination period of CNS development. Previously, we have identified a novel enhancer (designated ASE) in intron 1 DNA that appears to be important in mediating the surge of Plp1 gene activity during the active myelination period. Evidence suggests that the ASE participates in the formation of a specialized multi-protein/DNA complex called an enhanceosome. The current study describes an optimized, five-step, DNA affinity chromatography purification procedure to purify nuclear proteins from mouse brain that bind to the 85-bp ASE sequence, specifically. Electrophoretic mobility shift assay analysis demonstrated that specific DNA-binding activity was retained throughout the purification procedure, resulting in concomitant enrichment of nucleoprotein complexes. Identification of the purported regulatory factors was achieved through mass spectrometry analysis and included over 20 sequence-specific DNA-binding proteins. Supplementary western blot analyses to determine which of these sequence-specific factors are present in oligodendrocytes, and their developmental and regional expression in whole brain, suggest that Pur, and Pur, rank highest among the candidate factors as constituents of the multi-protein complex formed on the ASE. [source]

    Activated JNK brings about accelerated apoptosis of Bcl-2-overexpressing C6 glioma cells on treatment with tamoxifen

    JOURNAL OF NEUROCHEMISTRY, Issue 1 2005
    Madhavi S. Moodbidri
    Abstract Tamoxifen causes apoptosis of malignant glial cells at a concentration that does not kill normal astrocytes. C6 glioma cells were stably transfected with a vector expressing Bcl-2 under the control of metallothionin promoter. Low leaky Bcl-2 expression offered complete protection against tamoxifen-induced apoptosis. High Bcl-2 levels, on the other hand, accelerated the apoptosis, with Bcl-2-overexpressing clones dying within 48 h of tamoxifen treatment as compared to 6 days for parental C6 cells. Overexpressed Bcl-2 is localized primarily in mitochondria and to a much lower extent in endoplasmic reticulum (ER). Only a minor fraction of the overexpressed Bcl-2 gets phosphorylated in tamoxifen-treated cells and the phosphorylation does not affect its binding to Bax. Tamoxifen treatment of Bcl-2-overexpressing clones was found to result in activation of c-Jun N-terminal kinase (JNK) and p38 kinase. Inhibition of JNK but not p38 kinase completely abrogated the accelerated apoptosis. Constitutively expressed endogenous c-Jun was found to be phosphorylated, resulting in increased activator protein 1 (AP-1) DNA-binding activity. Expression of Fas ligand (FasL), an AP-1 transcriptional target, increased during accelerated cell death. This presumably brought about activation of caspase 8, as inhibition of caspase 8 blocked the apoptosis. The JNK/c-Jun/AP-1/FasL pathway could be considered as a potential target for the therapy of gliomas. [source]

    Heat Shock Transcription Factors and the hsp70 Induction Response in Brain and Kidney of the Hyperthermic Rat During Postnatal Development

    JOURNAL OF NEUROCHEMISTRY, Issue 1 2000
    Andrew J. Morrison
    Abstract : Heat shock transcription factor (HSF) 1 levels increase in brain regions and decline in kidney during postnatal rat development. In both neonatal and adult rats, levels of HSF1 protein in brain and kidney are proportional to the levels of HSF DNA-binding activity and the magnitude of heat shock protein hsp70 induction after thermal stress. There appears to be more HSF1 protein in adult brain than is needed for induction of hsp70 after thermal stress, suggesting that HSF1 may have other functions in addition to its role as a stress-inducible activator of heat shock genes. HSF2 protein levels decline during postnatal rat development in brain regions and kidney. Gel mobility shift analysis shows that HSF2 is not in a DNA-binding form in the neonatal brain and kidney, suggesting that HSF2 may not be involved in the constitutive expression of hsps in early postnatal development. There is no apparent relationship between levels of HSF2 protein and basal levels of hsp90, hsp70, heat shock cognate protein hsc70, and hsp60. [source]

    Ethanol Impairs Activation of Retinoic Acid Receptors in Cerebellar Granule Cells in a Rodent Model of Fetal Alcohol Spectrum Disorders

    ALCOHOLISM, Issue 5 2010
    Ambrish Kumar
    Background:, Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to ethanol toxic effects. Retinoic acid (RA) is a physiologically active metabolite of vitamin A that is locally synthesized in the cerebellum. Studies have shown that RA is required for neuronal development, but it remains unknown if ethanol impairs RA signaling and thus induces neuronal malformations. In this study, we tested the hypothesis that ethanol impairs the expression and activation of RA receptors in cerebellum and in cerebellar granule cells. Methods:, The cerebellum of ethanol unexposed and exposed pups was used to study the expression of retinoic acid receptors (RARs or RXRs) by immunohistochemistry and by Western blot analysis. We also studied the effect of ethanol on expression of RA receptors in the cerebellar granule cells. Activation of RA receptors (DNA-binding activities) in response to high-dose ethanol was determined by electrophoretic mobility shift and supershift assays. Results:, Findings from these studies demonstrated that ethanol exposure reduced the expression of RAR,/, while it increased the expression of RXR,/, in the cerebellum and in cerebellar granule neurons. Immuno-histological studies further strengthened the expression pattern of RA receptors in response to ethanol. The DNA-binding activity of RARs was reduced, while DNA-binding activity of RXRs was increased in response to ethanol exposure. Conclusion:, For the first time, our studies have demonstrated that high-dose ethanol affects the expression and activation of RA receptors, which could impair the signaling events and induce harmful effects on the survival and differentiation of cerebellar granule cells. Taken together, these findings could provide insight into the treatment options for brain defects caused by excessive ethanol exposure, such as in Fetal Alcohol Spectrum Disorders. [source]

    Inositol hexaphosphate downregulates both constitutive and ligand-induced mitogenic and cell survival signaling, and causes caspase-mediated apoptotic death of human prostate carcinoma PC-3 cells,

    MOLECULAR CARCINOGENESIS, Issue 1 2010
    Mallikarjuna Gu
    Abstract Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen-activated protein kinase (MAPK)- and Akt-mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF-1)-induced biological responses and associated signaling cascades in advanced and androgen-independent human PCa PC-3 cells. Treatment of PC-3 cells with 2,mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC-3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand-induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal-regulated kinase 1/2 (ERK1/2), c- Jun N-terminal protein kinase (JNK1/2), and p38 under constitutive and ligand-activated conditions. Under similar condition, IP6 also inhibited AP-1 DNA-binding activity and decreased nuclear levels of both phospho and total c-Fos and c- Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF-1 receptor (IGF-1R) pathway-mediated sustained growth promoting and survival signaling cascades in advanced and androgen-independent human PCa PC-3 cells, which might have translational implications in advanced human PCa control and management. © 2009 Wiley-Liss, Inc. [source]

    Involvement of Transcription Factor HNF3, in the Effect of o -Aminoazotoluene on Glucocorticoid Induction of Tyrosine Aminotransferase in Mice Sensitive to its Hepatocarcinogenic Action

    MOLECULAR CARCINOGENESIS, Issue 1 2001
    Konstantin Y. Kropachev
    Abstract In the rodent liver, hepatocarcinogens inhibit the glucocorticoid induction of several liver-specific genes, including tyrosine aminotransferase (TAT). A distinct positive correlation exists in mice between the extent of inhibition of TAT induction after acute administration of o -aminoazotoluene (OAT) and the frequency of liver tumors after chronic exposure to the carcinogen. To elucidate the mechanism of the carcinogenic action, the effects of OAT on the DNA-binding activity of several transcription factors participating in the glucocorticoid regulation of TAT gene expression were studied. The experimental inbred male mice were sensitive (A/He and SWR/J, tumor induction frequency of 75,100%, TAT induction inhibition of 35,50%) and resistant (CC57BR/Mv and AKR/J, 0,6% and 10,15%, respectively) to OAT. Gel retardation experiments showed that hepatocyte nuclear factor 3 (HNF3), DNA-binding activity was strongly reduced in nuclear extracts from the livers of OAT-treated A/He and SWR/J mice but only slightly reduced in CC57Br/Mv and AKR/J mice. The DNA-binding activities of Ets, AP1 family members, and GME binding proteins were unaffected. HNF3, DNA-binding activity was reduced by 1 h after OAT administration and remained low for 1 mo, as did inhibition of TAT induction in the liver. These results suggested that the inhibitory effect of OAT on the glucocorticoid induction of TAT is mediated by reduced HNF3, DNA-binding activity. © 2001 Wiley-Liss, Inc. [source]

    Temporal separation of distinct differentiation pathways by a dual specificity Rap-Phr system in Bacillus subtilis

    MOLECULAR MICROBIOLOGY, Issue 1 2007
    Wiep Klaas Smits
    Summary In bacterial differentiation, mechanisms have evolved to limit cells to a single developmental pathway. The establishment of genetic competence in Bacillus subtilis is controlled by a complex regulatory circuit that is highly interconnected with the developmental pathway for spore formation, and the two pathways appear to be mutually exclusive. Here we show by in vitro and in vivo analyses that a member of the Rap family of proteins, RapH, is activated directly by the late competence transcription factor ComK, and is capable of inhibiting both competence and sporulation. Importantly, RapH is the first member of the Rap family that demonstrates dual specificity, by dephosphorylating the Spo0F,P response regulator and inhibiting the DNA-binding activity of ComA. The protein thus acts at the stage where competence is well initiated, and prevents initiation of sporulation in competent cells as well as contributing to the escape from the competent state. A deletion of rapH induces both differentiation pathways and interferes with their temporal separation. Together, these results indicate that RapH is an integral part of a multifactorial regulatory circuit affecting the cell's decision between distinct developmental pathways. [source]

    The DNA-remodelling activity of DnaD is the sum of oligomerization and DNA-binding activities on separate domains

    MOLECULAR MICROBIOLOGY, Issue 4 2006
    Maria J. V. M. Carneiro
    Summary The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C-terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains. [source]

    Role of the N-terminal Region in the Function of the Photosynthetic Bacterium Transcription Regulator PpsR,

    PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
    Yoichi 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]

    A novel upstream regulator of WRKY53 transcription during leaf senescence in Arabidopsis thaliana

    PLANT BIOLOGY, Issue 2008
    Y. Miao
    Abstract Arabidopsis WRKY proteins comprise a family of zinc finger-type transcription factors involved in the regulation of gene expression during pathogen defence, wounding, trichome development and senescence. To better understand the regulatory role of the senescence-related WRKY53 factor, we identified upstream regulatory factors using the yeast one-hybrid system. Among others, we identified a DNA-binding protein with a so far unknown function that contains a transcriptional activation domain and a kinase domain with similarities to HPT kinases. In vitro studies revealed that this activation domain protein (AD protein) can phosphorylate itself and that phosphorylation increases its DNA-binding activity to the WRKY53 promoter region. Using the yeast two-hybrid system, an interaction with proteins that were previously shown to bind to the WRKY53 promoter was tested. The AD protein interacted with MEKK1. The interaction with MEKK1 was confirmed in vivo by bimolecular fluorescence complementation (BiFC); however, the AD protein was not phosphorylated by MEKK1 in vitro and vice versa. This indicates that there may be competition between WRKY53 and AD protein for binding of MEKK1 at the WRKY53 promoter. Overexpression and knockout of the respective gene resulted in changes in transcription levels of WRKY53, indicating that AD protein is a positive regulator of WRKY53 expression. Expression of the AD protein gene can be induced by hydrogen peroxide treatment and reduced by jasmonic acid treatment, as previously shown for WRKY53. [source]

    The solution structure of ZNF593 from Homo sapiens reveals a zinc finger in a predominately unstructured protein

    PROTEIN SCIENCE, Issue 3 2008
    Paulette L. Hayes
    Abstract Here, we report the solution structure of ZNF593, a protein identified in a functional study as a negative modulator of the DNA-binding activity of the Oct-2 transcription factor. ZNF593 contains a classic C2H2 zinc finger domain flanked by about 40 disordered residues on each terminus. Although the protein contains a high degree of intrinsic disorder, the structure of the zinc finger domain was resolved by NMR spectroscopy without a need for N- or C-terminal truncations. The tertiary structure of the zinc finger domain is composed of a ,-hairpin that positions the cysteine side chains for zinc coordination, followed by an atypical kinked ,-helix containing the two histidine side chain ligands. The structural topology of ZNF593 is similar to a fragment of the double-stranded RNA-binding protein Zfa and the C-terminal zinc finger of MBP-1, a human enhancer binding protein. The structure presented here will provide a guide for future functional studies of how ZNF593 negatively modulates the DNA-binding activity of Oct-2, a POU domain-containing transcription factor. Our work illustrates the unique capacity of NMR spectroscopy for structural analysis of folded domains in a predominantly disordered protein. [source]

    The redox-sensing regulator YodB senses quinones and diamide via a thiol-disulfide switch in Bacillus subtilis

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2010
    Bui Khanh Chi
    Abstract The MarR/DUF24-type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox-sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6-Ala mutant that the conserved Cys6 apparently contributes to the DNA-binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox-sensing in response to diamide and 2-methylhydroquinone (MHQ) in vivo. DNA-binding experiments indicate that YodB is regulated by a reversible thiol-modification in response to diamide and MHQ in vitro. Redox-regulation of YodB involves Cys6-Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C-terminal Cys101' or Cys108' residues. This study reveals a thiol-disulfide switch model of redox-regulation for the YodB repressor to sense electrophilic compounds in vivo. [source]