C/EBP DNA-binding activity is upregulated by a glucocorticoid-dependent mechanism in septic muscle

2002 ◽  
Vol 282 (2) ◽  
pp. R439-R444 ◽  
Author(s):  
Gail Penner ◽  
Gyu Gang ◽  
Xiaoyan Sun ◽  
Curtis Wray ◽  
Per-Olof Hasselgren
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factors ◽  
Dna Binding ◽  
Cecal Ligation ◽  
Binding Activity ◽  
Nuclear Fraction ◽  
Mobility Shift ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Mobility Shift Assay

Sepsis-induced muscle cachexia is associated with increased expression of several genes in the ubiquitin-proteasome proteolytic pathway, but little is known about the activation of transcription factors in skeletal muscle during sepsis. We tested the hypothesis that sepsis upregulates the expression and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)-β and -δ in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture, and control rats were sham operated. C/EBP-β and -δ DNA-binding activity was determined by electrophoretic mobility shift assay and supershift analysis. In addition, C/EBP-β and -δ nuclear protein levels were determined by Western blot analysis. Sepsis resulted in increased DNA-binding activity of C/EBP, and supershift analysis suggested that this reflected activation of the β- and δ-isoforms of C/EBP. Concomitantly, C/EBP-β and -δ protein levels were increased in the nuclear fraction of skeletal muscle. In additional experiments, we tested the role of glucocorticoids in sepsis-induced activation of C/EBP-β and -δ by treating rats with the glucocorticoid receptor antagonist RU-38486. This treatment inhibited the sepsis-induced activation of C/EBP-β and -δ, suggesting that glucocorticoids participate in the upregulation of C/EBP in skeletal muscle during sepsis. The present results suggest that C/EBP-β and -δ are activated in skeletal muscle during sepsis and that this response is, at least in part, regulated by glucocorticoids.

Examination of DNA-binding activity of neuronal transcription factors by electrophoretical mobility shift assay

1998 ◽  
Vol 2 (4) ◽  
pp. 243-249 ◽  
Author(s):  
Koichiro Kako ◽  
Hisanori Wakamatsu ◽  
Toshiyuki Hamada ◽  
Marek Banasik ◽  
Keiko Ohata ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Activity ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Mobility Shift Assay

scholarly journals DNA-binding activity in the excretory–secretory products ofTrichinella pseudospiralis(Nematoda: Trichinelloidea)

Parasitology ◽  
2001 ◽  
Vol 123 (3) ◽  
pp. 301-308 ◽  
Author(s):  
C. H. MAK ◽  
R. C. KO
Keyword(s):  
Dna Binding ◽  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Binding Activity ◽  
Mobility Shift ◽  
Competition Analysis ◽  
Dna Binding Activity ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Secretory Products

A novel DNA-binding peptide ofMr∼30 kDa was documented for the first time in the excretory–secretory (E–S) products of the infective-stage larvae ofTrichinella pseudospiralis.Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E–S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of theMrof proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E–S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E–S product.

scholarly journals The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

1994 ◽  
Vol 14 (7) ◽  
pp. 4380-4389 ◽  
Author(s):  
L I Chen ◽  
T Nishinaka ◽  
K Kwan ◽  
I Kitabayashi ◽  
K Yokoyama ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Product ◽  
Binding Activity ◽  
Negative Regulator ◽  
Control Element ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Extracts ◽  
Mobility Shift Assay

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

Determination of DNA Binding Behavior of FoxA1 Constructs Using a Gold Nanoparticle-Based High Throughput Assay

2016 ◽  
Vol 04 (03) ◽  
pp. 1640012
Author(s):  
Khin Moh Moh Aung ◽  
Michelle Gek Liang Lim ◽  
Shuzhen Hong ◽  
Edwin Cheung ◽  
Xiaodi Su
Keyword(s):  
Dna Binding ◽  
Screening Method ◽  
Binding Activity ◽  
Mobility Shift ◽  
Molecular Weights ◽  
Binding Behavior ◽  
Dna Binding Activity ◽  
Multiple Organs ◽  
Mobility Shift Assay ◽  
High Throughput Assay

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged-helix transcription factors. It plays crucial roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. In this study, in order to determine the regions of FoxA1 necessary for efficient Deoxyribonucleic Acid (DNA) binding, we cloned, expressed and purified a series of FoxA1 constructs that contain either the DNA Binding Domain (DBD), the Transcription Activation Domain (TAD), or both. We determined the DNA binding behavior of these constructs using traditional electrophoretic mobility shift assay (EMSA) and a recently developed gold nanoparticles (AuNPs)-based fast screening method. We conclude that just the DBD region alone is not sufficient for protein-DNA binding activity. Amino acids flanking the upstream of the DBD region are required for maximal DNA binding activity. Through this study, we have also further validated the AuNPs assay for its generality and expanded the existing protocol for comparing the DNA binding behavior of multiple proteins of different charge properties and molecular weights.

Creatine feeding increases GLUT4 expression in rat skeletal muscle

2005 ◽  
Vol 288 (2) ◽  
pp. E347-E352 ◽  
Author(s):  
Jeong-Sun Ju ◽  
Jill L. Smith ◽  
Peter J. Oppelt ◽  
Jonathan S. Fisher
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Dna Binding ◽  
Binding Activity ◽  
Rat Skeletal Muscle ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Glut4 Expression ◽  
Edl Muscle ◽  
Glut4 Gene Expression

The purpose of this study was to investigate the potential role of creatine in GLUT4 gene expression in rat skeletal muscle. Female Wistar rats were fed normal rat chow (controls) or chow containing 2% creatine monohydrate ad libitum for 3 wk. GLUT4 protein levels of creatine-fed rats were significantly increased in extensor digitorum longus (EDL), triceps, and epitrochlearis muscles compared with muscles from controls ( P < 0.05), and triceps GLUT4 mRNA levels were ∼100% greater in triceps muscles from creatine-fed rats than in muscles from controls ( P < 0.05). In epitrochlearis muscles from creatine-fed animals, glycogen content was ∼40% greater ( P < 0.05), and insulin-stimulated glucose transport rates were higher ( P < 0.05) than in epitrochlearis muscles from controls. Despite no changes in [ATP], [creatine], [phosphocreatine], or [AMP], creatine feeding increased AMP-activated protein kinase (AMPK) phosphorylation by 50% in rat EDL muscle ( P < 0.05). Creatinine content of EDL muscle was almost twofold higher for creatine-fed animals than for controls ( P < 0.05). Creatine feeding increased protein levels of myocyte enhancer factor 2 (MEF2) isoforms MEF2A (∼70%, P < 0.05), MEF2C (∼60%, P < 0.05), and MEF2D (∼90%, P < 0.05), which are transcription factors that regulate GLUT4 expression, in creatine-fed rat EDL muscle nuclear extracts. Electrophoretic mobility shift assay showed that DNA binding activity of MEF2 was increased by ∼40% ( P < 0.05) in creatine-fed rat EDL compared with controls. Our data suggest that creatine feeding enhances the nuclear content and DNA binding activity of MEF2 isoforms, which is concomitant with an increase in GLUT4 gene expression.

CRTF is a novel transcription factor that regulates multiple stages of Dictyostelium development

Development ◽  
2001 ◽  
Vol 128 (13) ◽  
pp. 2569-2579 ◽  
Author(s):  
Xiuqian Mu ◽  
Seth A. Spanos ◽  
Joseph Shiloach ◽  
Alan Kimmel
Keyword(s):  
Dna Binding ◽  
Constitutive Expression ◽  
Binding Activity ◽  
Receptor Subtype ◽  
Continuous Exposure ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Expression Pathways ◽  
Mobility Shift Assay ◽  
Camp Receptor

During aggregation, Dictyostelium establish nanomolar oscillation waves of extracellular cAMP, but as development progresses, cells become responsive to higher, non-fluctuating concentrations of cAMP. The regulation of the promoter responsible for expression of cAMP receptor subtype 1, CAR1, during aggregation reflects these signaling variations. Transcription of CAR1 from the early, aggregation promoter is activated by cAMP pulsing, but is repressed by continuous exposure to micromolar concentrations of cAMP. Deletion and mutation analyses of this promoter had defined an element essential for cAMP-regulated expression, and mobility shift assay, DNA crosslinking and DNase I footprinting experiments had identified a nuclear protein (CRTF) with zinc-dependent sequence binding specificity. In our study, CRTF was purified to homogeneity, peptides were sequenced and full-length cDNAs were obtained. The deduced CRTF protein is ∼100 kDa with a C-terminal, zinc finger-like motif required for DNA binding; CRTF purified from cells, however, represents only a 40 kDa C-terminal fragment that retains DNA-binding activity. As might have been predicted if CRTF were essential for the regulation of CAR1, crtf-null strains fail to develop under standard conditions or to exhibit induced expression of CAR1 or other cAMP-regulated genes. Furthermore, crtf-nulls also fail to sporulate, even under conditions that bypass the dependence on early cAMP signaling pathways. In addition, early developmental events of crtf-null strains could be rescued with exogenous cAMP treatment, constitutive expression of CAR1 or co-development with wild-type cells; however, these treatments were insufficient to promote sporulation. This suggests a cell-autonomous role for CRTF during late development that is separate from its capacity to control CAR1 expression. Finally, ablation of CRTF promotes a precocious induction of certain cAMP-dependent gene expression pathways. We suggest that CRTF may function to help insulate distinct pathways from simultaneous and universal activation by cAMP. CRTF, thus, exhibits multiple complex and independent regulatory functions during Dictyostelium development.

EPA inhibits the inhibitor of κBα (IκBα)/NF-κB/muscle RING finger 1 pathway in C2C12 myotubes in a PPARγ-dependent manner

2010 ◽  
Vol 105 (3) ◽  
pp. 348-356 ◽  
Author(s):  
Feiruo Huang ◽  
Hongkui Wei ◽  
Hefeng Luo ◽  
Siwen Jiang ◽  
Jian Peng
Keyword(s):  
Dna Binding ◽  
Mrna Expression ◽  
Ring Finger ◽  
Binding Activity ◽  
C2c12 Myotubes ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Fold Reduction ◽  
Mobility Shift Assay

The present study was conducted to evaluate the mechanism by which n-3 PUFA regulates the inhibitor of κBα (IκBα)/NF-κB/muscle RING finger 1 (MuRF1) pathway in C2C12 myotubes. After treatment with 150, 300 or 600 μm-α-linolenic acid (ALA) or -EPA for 24 h in C2C12 myotubes, the levels of phosphorylated IκBα (p-IκBα) and total IκBα were measured by Western blot. Compared with the bovine serum albumin (BSA) control, 150 and 300 μm-ALA and -EPA, respectively, did not affect the total IκBα protein level (P>0·05). However, 600 μm-EPA, but not 600 μm-ALA, prevented IκBα phosphorylation and increased the total IκBα levels (P < 0·01). Furthermore, total nuclear protein was isolated and analysed by the electrophoretic mobility shift assay for NF-κB DNA-binding activity after treatment with 600 μm-ALA or -EPA for 24 h. EPA (600 μm), but not ALA (600 μm), decreased the NF-κB DNA-binding activity when compared with BSA (P < 0·01). It was further observed that 600 μm-EPA caused a 3·38-fold reduction in the levels of MuRF1 mRNA expression compared with BSA (P < 0·01). Additionally, 600 μm-EPA resulted in a 2·3-fold induction of PPARγ mRNA expression (P < 0·01). In C2C12 myotubes, PPARγ knockdown by RNA interference significantly decreased PPARγ mRNA and protein expression to approximately 50 and 60 % (P < 0·01), respectively. Interestingly, in C2C12 myotubes with PPARγ knockdown, 600 μm-ALA and -EPA did not affect the levels of p-IκBα and total IκBα, NF-κB DNA-binding activity or MuRF1 mRNA expression when compared with BSA (P>0·05). These results revealed that EPA, but not ALA, inhibited the IκBα/NF-κB/MuRF1 pathway in C2C12 myotubes in a PPARγ-dependent manner.

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