scholarly journals The NF-κB p50 Subunit Is Protective during Intestinal Entamoeba histolytica Infection of 129 and C57BL/6 Mice

2010 ◽  
Vol 78 (4) ◽  
pp. 1475-1481 ◽  
Author(s):  
Kyou-Nam Cho ◽  
Stephen M. Becker ◽  
Eric R. Houpt
Keyword(s):  
Entamoeba Histolytica ◽  
Electrical Resistance ◽  
Transepithelial Electrical Resistance ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mobility Shift ◽  
Protective Function ◽  
Targeted Deletion ◽  
Nuclear Extracts ◽  
Histolytica Infection ◽  
Mobility Shift Assay

ABSTRACT Entamoeba histolytica is the agent of amebic colitis. In this work we examined the intestinal NF-κB response to this parasite. Using an enzyme-linked immunosorbent assay (ELISA) and an electrophoretic mobility shift assay, we found that the NF-κB subunit p50 predominated in nuclear extracts of whole cecal tissue and of isolated crypts from mice inoculated with E. histolytica. p50 was protective, since C57BL/6 and 129 mice in which there was targeted deletion of this subunit were more susceptible to E. histolytica infection as measured by culture results, cecal parasite ELISA results, and/or histologic scores. The transepithelial electrical resistance of cecal explants from C57BL/6 and 129 p50 knockout mice decreased markedly in response to the parasite compared with the transepithelial electrical resistance of their wild-type counterparts, suggesting that a protective function of p50 was present in the epithelium itself. This work shows that NF-κB activity, particularly activity of the p50 subunit, is one factor that contributes to resistance of the gut to E. histolytica infection.

scholarly journals Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

2005 ◽  
Vol 70 (5) ◽  
pp. 705-712 ◽  
Author(s):  
Miroslava Vujcic ◽  
Natasa Terzic ◽  
Aleksandra Ristic-Fira ◽  
Dusan Kanazir ◽  
Sabera Ruzdijic
Keyword(s):  
Dna Interaction ◽  
Binding Activity ◽  
Regulatory Proteins ◽  
Nuclear Proteins ◽  
Middle Aged ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Oligonucleotide Analogues

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. .

scholarly journals Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

2019 ◽  
Vol 476 (8) ◽  
pp. 1247-1266
Author(s):  
Zhanjun Hou ◽  
Carrie O'Connor ◽  
Josephine Frühauf ◽  
Steve Orr ◽  
Seongho Kim ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Transcriptional Control ◽  
Consensus Sequence ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Ht1080 Cells ◽  
Promoter Construct ◽  
Mobility Shift Assay ◽  
Reporter Gene Assays

Abstract Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94. Progressive deletions of the hPCFT promoter construct (−2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions −50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.

scholarly journals A Sequence of the CIS Gene Promoter Interacts Preferentially with Two Associated STAT5A Dimers: a Distinct Biochemical Difference between STAT5A and STAT5B

1998 ◽  
Vol 18 (10) ◽  
pp. 5852-5860 ◽  
Author(s):  
Frédérique Verdier ◽  
Raquel Rabionet ◽  
Fabrice Gouilleux ◽  
Christian Beisenherz-Huss ◽  
Paule Varlet ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Prolactin Receptor ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Binding Sequence ◽  
Biochemical Difference

ABSTRACT Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.

scholarly journals Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.

1993 ◽  
Vol 178 (5) ◽  
pp. 1681-1692 ◽  
Author(s):  
L R Gottschalk ◽  
D M Giannola ◽  
S G Emerson
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Protein Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Nuclear Protein ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to &lt; 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells

2008 ◽  
Vol 416 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Manjunath B. Joshi ◽  
Danila Ivanov ◽  
Maria Philippova ◽  
Emmanouil Kyriakakis ◽  
Paul Erne ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Protein Levels ◽  
Nuclear Extracts ◽  
Nucleoprotein Complex ◽  
Thioredoxin 1 ◽  
Mobility Shift Assay ◽  
Reporter Gene Analysis

T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H2O2) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from −99 to −2304 bp, located the minimal promoter region of T-cad within −285 bp from the translation start site. Reporter activity in ECs transfected with the −285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to −156 to −203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to −156 to −203 bp after streptavidin–agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.

Structural Analysis and Identification of Cis-Elements of Rice osRACD Gene

2004 ◽  
Vol 36 (3) ◽  
pp. 191-198 ◽  
Author(s):  
Yun-Zhe Xu ◽  
Rui-Lin You ◽  
Nai-Hu Wu
Keyword(s):  
Oryza Sativa L ◽  
Small Gtpase ◽  
Inverse Pcr ◽  
Upstream Region ◽  
Male Sterile ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Photoperiod Sensitive ◽  
Gel Mobility Shift

Abstract The osRACD gene correlated with fertility transformation in the photoperiod sensitive genic male sterile rice (PGMR), Nongken 58S, encoded a rice (Oryza sativa L. ssp. japonica) small GTPase belonging to the Rac/Rho family. Inverse PCR was performed to amplify a fragment about 1.4 kb in 5′ upstream region of the osRACD promoter. Deletion mutation and gel mobility shift assay characterized two fragments (–799 to –686 nt, and –686 to –431 nt) in the osRACD promoter that could be involved in its transcriptional regulation. When these two deletion fragments were used as probe respectively, a retarded band appeared in the nuclear extracts of fertile 58S rice under short day (58S-SD). Whereas no retarded band was shown in the nuclear extracts of sterile 58S rice under long day (58S-LD). Competition assay indicated that the factors in the retarded bands binding to these two fragments were the same trans-acting factor (termed rice factor, RF). The binding affinity of RF was affected by phosphorylation and was higher in SD-growth rice than that of LD-growth rice.

scholarly journals Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Nature ◽  
2022 ◽  
Author(s):  
Shikang Liang ◽  
Sherine E. Thomas ◽  
Amanda K. Chaplin ◽  
Steven W. Hardwick ◽  
Dimitri Y. Chirgadze ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Inhibitory Effect ◽  
Rational Drug Design ◽  
Dna Double Strand Breaks ◽  
Mobility Shift ◽  
Dependent Protein Kinase ◽  
Drug Candidates ◽  
Nuclear Extracts ◽  
Dependent Protein ◽  
Mobility Shift Assay

AbstractThe DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

Increased contractile activity decreases RNA-protein interaction in the 3'-UTR of cytochrome c mRNA

1996 ◽  
Vol 271 (4) ◽  
pp. C1157-C1166 ◽  
Author(s):  
Z. Yan ◽  
S. Salmons ◽  
Y. I. Dang ◽  
M. T. Hamilton ◽  
F. W. Booth
Keyword(s):  
Cytochrome C ◽  
Protein Interaction ◽  
Tibialis Anterior ◽  
Contractile Activity ◽  
Supernatant Fraction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mobility Shift ◽  
Muscle Extract ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

This study was designed to gain an insight into mechanisms by which cytochrome c gene expression is enhanced by increased contractile activity in skeletal muscle. When rat tibialis anterior muscles were stimulated (10 Hz, 0.25 ms) for 0, 2, 6, 12, or 24 h or 2, 5, 9, or 13 days (n = 4 for each time point), cytochrome c protein (enzyme-linked immunosorbent assay) and mRNA (Northern blot analysis) concentrations started to increase by 9 days, and this was associated with concurrent decreases in cytochrome c mRNA-protein interaction (RNA gel mobility shift assay). We found that the decreased RNA-protein interaction in the stimulated muscle extract was restored by ultracentrifugation (150,000 g, 1 h) in the supernatant fraction. The 150,000 g pellet fraction of stimulated muscle was capable of inhibiting the RNA-protein interaction in control tibialis anterior muscles. These results provide evidence of an inhibitory factor that is responsible for decreasing RNA-protein interaction in the 3'-untranslated region of cytochrome c mRNA in continuously stimulated muscle.

An upstream element of the TamS1 gene is a site of DNA-protein interactions during differentiation to the merozoite in Theileria annulata

2000 ◽  
Vol 113 (12) ◽  
pp. 2243-2252 ◽  
Author(s):  
B. Shiels ◽  
M. Fox ◽  
S. McKellar ◽  
J. Kinnaird ◽  
D. Swan
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Theileria Annulata ◽  
Upstream Region ◽  
Fundamental Aspect ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
High Level

Apicomplexan parasites are major pathogens of humans and domesticated animals. A fundamental aspect of apicomplexan biology, which may provide novel molecular targets for parasite control, is the regulation of stage differentiation. Studies carried out on Theileria annulata, a bovine apicomplexan parasite, have provided evidence that a stochastic process controls differentiation from the macroschizont to the merozoite stage. It was postulated that this process involves the presence of regulators of merozoite gene expression in the preceding stage of the life cycle, and that during differentiation a quantitative increase of these factors occurs. This study was carried out to test these postulations. Nuclear run-on analysis showed that TamS1 expression is controlled, at least in part, at the transcriptional level. The transcription start site showed homology with the consensus eukaryotic initiator motif, and study of the 5′ upstream region by the electrophoretic mobility-shift assay demonstrated that a 23 bp motif specifically bound factors from parasite-enriched nuclear extracts. Three complexes were shown to bind to a 9 bp core binding site (5′-TTTGTAGGG-3′). Two of these complexes were present in macroschizont extracts but were found at elevated levels during differentiation. Both complexes contain a polypeptide of the same molecular mass and may be related via the formation of homodimer or heterodimer complexes. The third complex appears to be distinct and was detected at time points associated with the transition to high level merozoite gene expression.

Comparison of Two Different Techniques to Assess Adalimumab Trough Levels in Patients with Crohn’s Disease

2015 ◽  
Vol 24 (4) ◽  
pp. 451-456 ◽  
Author(s):  
Giorgia Bodini ◽  
Vincenzo Savarino ◽  
Edoardo G. Giannini ◽  
Manuele Furnari ◽  
Elisa Marabotto ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Tumor Necrosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mobility Shift ◽  
Loss Of Response ◽  
Inflammatory Bowel ◽  
Mobility Shift Assay ◽  
Trough Levels ◽  
Necrosis Factor

Background & Aims:  Loss of response to anti-tumor necrosis factor (TNF) drugs in patients with inflammatory bowel disease is likely due to low drug serum levels, and dosing anti-TNF drug concentrations may improve patients’ outcome. However, there are limited data on the diagnostic accuracy and utility of currently available assays for measuring anti-TNF levels. In this study, our aim was to compare serum adalimumab concentrations with two different techniques. Methods: We assessed serum adalimumab concentrations in 23 patients with Crohn’s disease during a 96-week follow-up period. Adalimumab trough levels were assessed using a sandwich principle-based enzyme-linked immunosorbent assay (ELISA) and a homogeneous mobility shift assay (HMSA). Results: At week 48, adalimumab trough levels were significantly lower in patients who experienced relapse compared to patients in remission, using both ELISA and HMSA methods: 4.8 mcg/mL (2.4-7.2 mcg/mL) vs. 7.5 mcg/mL (6.6-8.4 mcg/mL) (P=0.01) and 6.5 mcg/mL (3-10 mcg/mL) vs. 11.6 mcg/mL (7-16.2 mcg/ml) (P=0.004), respectively. Similar results were obtained at week 96: 5.9 mcg/mL (3.3-8.5 mcg/mL) vs. 12.8 mcg/mL (9.4-16.2 mcg/mL) (P=0.001) and 4.1 mcg/mL (1.6-6.6 mcg/mL) vs. 7.5 mcg/mL (5.7-9.3 mcg/mL) (P=0.009), respectively. There was a significant correlation between ELISA and HMSA adalimumab trough levels at both 48 (r = 0.691, P=0.0003) and 96 week (r = 0.822, P=0.0001). Conclusions: ELISA and HMSA assays are accurate methods to assess adalimumab trough levels in patients with Crohn’s disease and those who experience loss of response. The preferential use of one of the two techniques should be based on local availability and physicians’ experience.Abbreviations: ADA: Adalimumab; AA: Anti-drug antibodies; anti-TNF: Anti-tumor necrosis factor; CD: Crohn’s disease; ELISA: Enzyme-linked immunosorbent assay; HBI: Harvey-Bradshaw Index; HMSA: Homogeneous mobility shift assay; IBD: Inflammatory bowel diseases; LOR: Loss of response.

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