scholarly journals Interleukin-10-mediated inhibition of free radical generation in macrophages

2001 ◽  
Vol 280 (6) ◽  
pp. L1196-L1202 ◽  
Author(s):  
Sujatha Dokka ◽  
Xianglin Shi ◽  
Stephen Leonard ◽  
Liying Wang ◽  
Vincent Castranova ◽  
...  
Keyword(s):  
Gene Transfection ◽  
Sodium Formate ◽  
Nuclear Factor Κb ◽  
Interleukin 10 ◽  
Oh Radicals ◽  
Mobility Shift ◽  
Inflammatory Processes ◽  
Mobility Shift Assay ◽  
Metal Catalyzed

Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines that are known to be transcriptionally regulated by nuclear factor-κB (NF-κB). Although still controversial, IL-10 has been shown to inhibit NF-κB activation through a process that involves proteolytic degradation of inhibitory subunit IκB-α. What is not known, however, is the mechanism by which IL-10 exerts its effect on IκB-α degradation. The present study investigates the possible role of reactive oxygen species (ROS) and their inhibition by IL-10 in NF-κB activation and IκB-α degradation in macrophages. Treatment of the cells with lipopolysaccharide (LPS) caused activation of NF-κB and rapid proteolysis of IκB-α as determined by the electrophoretic mobility shift assay, gene transfection, and Western blot. IL-10 pretreatment inhibited both NF-κB activation and IκB-α degradation. Both of these processes were also inhibited by ROS scavengers, catalase (H2O2 scavenger), and sodium formate (·OH scavenger) but were minimally affected by superoxide dismutase (O[Formula: see text] scavenger). These results suggests that ·OH radicals, formed by an H2O2-dependent, metal-catalyzed Fenton reaction, play a major role in this process. Electron spin resonance studies confirmed the formation of ·OH radicals in LPS-treated cells. Addition of IL-10 inhibited both IκB-α degradation and generation of ·OH radicals in response to LPS stimulation. These results demonstrate, for the first time, direct evidence for the role of IL-10 in ROS-dependent NF-κB activation.

Role of NF-κB in TNF-α-induced COX-2 Expression in Synovial Fibroblasts from Human TMJ

2007 ◽  
Vol 86 (4) ◽  
pp. 363-367 ◽  
Author(s):  
J. Ke ◽  
X. Long ◽  
Y. Liu ◽  
Y.F. Zhang ◽  
J. Li ◽  
...  
Keyword(s):  
Synovial Fibroblasts ◽  
Nuclear Factor Κb ◽  
Mobility Shift ◽  
Tnf Α ◽  
Cox 2 ◽  
Mobility Shift Assay ◽  
Pre Treatment ◽  
Factor Α ◽  
Necrosis Factor

In the temporomandibular joint (TMJ) synovium, cyclo-oxygenase-2 (COX-2) expression has been believed to be directly related to joint pain and synovitis. Here we investigated the role of Nuclear Factor κB (NF-κB) in the regulation of COX-2 expression in synovial fibroblasts from human TMJ induced by tumor necrosis factor-α (TNF-α). By reverse-transcriptase/polymerase chain-reaction (RT-PCR) and Western blotting analysis, TNF-α induced a dose- and time-dependent increase in COX-2 expression. Electrophoretic mobility shift assay (EMSA) revealed that transient NF-κB activation in the COX-2 promoter was triggered by TNF-α. In parallel with transient NF-κB activation, the rapid translocation of NF-κB, particularly the p65 subunit, from the cytoplasm into the nucleus was demonstrated. Pre-treatment with pyrolidine dithiocarbamate (PDTC), one of the NF-κB inhibitors, prevented binding to the COX-2 promoter and expression of COX-2 protein in response to TNF-α. These findings indicate that activation of NF-κB is responsible for TNF-α-induced COX-2 expression in synovial fibroblasts from the TMJ.

scholarly journals Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β promoter

2008 ◽  
Vol 42 (3) ◽  
pp. 225-237 ◽  
Author(s):  
Yumiko Kashiwabara ◽  
Shigekazu Sasaki ◽  
Akio Matsushita ◽  
Koji Nagayama ◽  
Kenji Ohba ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Minimal Sequence ◽  
Mobility Shift Assay ◽  
Β Chain

Thyrotropin (TSH) is a heterodimer consisting of α and β chains, and the β chain (TSHβ) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHβ expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHβ promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHβ promoter without PIT1. The deleted region (nt −82/−52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHβ promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

scholarly journals Ouabain Modulates Zymosan-Induced Peritonitis in Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Jacqueline Alves Leite ◽  
Anne Kaliery De Abreu Alves ◽  
José Guilherme Marques Galvão ◽  
Mariana Pires Teixeira ◽  
Luiz Henrique Agra Cavalcante-Silva ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Potent Inhibitor ◽  
Binding Activity ◽  
Acute Inflammatory Response ◽  
Mobility Shift ◽  
Plasma Exudation ◽  
Mobility Shift Assay ◽  
Number Of Cells

Ouabain, a potent inhibitor of the Na+, K+-ATPase, was identified as an endogenous substance. Recently, ouabain was shown to affect various immunological processes. We have previously demonstrated the ability of ouabain to modulate inflammation, but little is known about the mechanisms involved. Thus, the aim of the present work is to evaluate the immune modulatory role of ouabain on zymosan-induced peritonitis in mice. Our results show that ouabain decreased plasma exudation (33%). After induction of inflammation, OUA treatment led to a 46% reduction in the total number of cells, as a reflex of a decrease of polymorphonuclear leukocytes, which does not appear to be due to cell death. Furthermore, OUA decreased TNF-α(57%) and IL-1β(58%) levels, without interfering with IL-6 and IL-10. Also,in vitroexperiments show that ouabain did not affect endocytic capacity. Moreover, electrophoretic mobility shift assay (EMSA) shows that zymosan treatment increased (85%) NF-κB binding activity and that ouabain reduced (30%) NF-κB binding activity induced by zymosan. Therefore, our data suggest that ouabain modulated acute inflammatory response, reducing the number of cells and cytokines levels in the peritoneal cavity, as well as NFκB activation, suggesting a new mode of action of this substance.

scholarly journals DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis

2004 ◽  
Vol 186 (18) ◽  
pp. 6003-6014 ◽  
Author(s):  
Giuseppe Amati ◽  
Paola Bisicchia ◽  
Alessandro Galizzi
Keyword(s):  
Bacillus Subtilis ◽  
Adaptive Strategies ◽  
Promoter Sequence ◽  
Two Component System ◽  
Mobility Shift ◽  
Flagellin Gene ◽  
Transcriptional Termination ◽  
Phosphorylated Form ◽  
Mobility Shift Assay

ABSTRACT Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the σA-dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the PA promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.

scholarly journals Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

2006 ◽  
Vol 396 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Ferenc Marincs ◽  
Iain W. Manfield ◽  
Jonathan A. Stead ◽  
Kenneth J. Mcdowall ◽  
Peter G. Stockley
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Dna Arrays ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Metabolic Role ◽  
Mobility Shift Assay

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5′ promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein–protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein–protein and protein–DNA affinity.

scholarly journals Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods

2020 ◽  
Vol 21 (23) ◽  
pp. 9150
Author(s):  
Yoshitomo Shiroma ◽  
Go Fujita ◽  
Takuya Yamamoto ◽  
Ryou-u Takahashi ◽  
Ashutosh Kumar ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclear Factor Κb ◽  
Screening Methods ◽  
Biological Functions ◽  
Mobility Shift ◽  
Dna Strand Exchange ◽  
Mobility Shift Assay ◽  
Dna Strand

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA–DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

IL-1β induces eotaxin gene transcription in A549 airway epithelial cells through NF-κB

2000 ◽  
Vol 279 (6) ◽  
pp. L1058-L1065 ◽  
Author(s):  
Sean Jedrzkiewicz ◽  
Hidetoshi Nakamura ◽  
Eric S. Silverman ◽  
Andrew D. Luster ◽  
Naresh Mansharamani ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transcriptional Activation ◽  
Transcriptional Start Site ◽  
Nuclear Factor Κb ◽  
Airway Epithelial Cells ◽  
Site Directed Mutagenesis ◽  
Airway Epithelial ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Necessary And Sufficient

Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1β (IL-1β). We detected an increase in newly transcribed eotaxin mRNA in IL-1β-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1β-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-κB (NF-κB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1β induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1β-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-κB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-κB site ( r 2 = 0.97, P = 0.002). Moreover, IL-1β-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-κB. Our findings implicate NF-κB and its binding sequence in IL-1β-induced transcriptional activation of the eotaxin gene.

scholarly journals Cyclolinteinone, a sesterterpene from sponge Cacospongia linteiformis, prevents inducible nitric oxide synthase and inducible cyclo-oxygenase protein expression by blocking nuclear factor-κB activation in J774 macrophages

2000 ◽  
Vol 346 (3) ◽  
pp. 793-798 ◽  
Author(s):  
Fulvio D'ACQUISTO ◽  
Virginia LANZOTTI ◽  
Rosa CARNUCCIO
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Nuclear Translocation ◽  
Inflammatory Diseases ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
J774 Cells ◽  
Cox 2 ◽  
Mobility Shift Assay

We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 μg/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 μM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-ĸB (NF-ĸB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of IĸB-α from cytosolic fraction and nuclear translocation of NF-ĸB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-ĸB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.

scholarly journals The NtrC Family Regulator AlgB, Which Controls Alginate Biosynthesis in Mucoid Pseudomonas aeruginosa, Binds Directly to the algD Promoter

2007 ◽  
Vol 190 (2) ◽  
pp. 581-589 ◽  
Author(s):  
Andrew J. Leech ◽  
April Sprinkle ◽  
Lynn Wood ◽  
Daniel J. Wozniak ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Mobility Shift ◽  
Alginate Production ◽  
Mobility Shift Assay ◽  
Exponential Enrichment ◽  
Microarray Analyses ◽  
Phosphorylation Domain

ABSTRACT Alginate production in mucoid (MucA-defective) Pseudomonas aeruginosa is dependent upon several transcriptional regulators, including AlgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, KinB, and even the N-terminal phosphorylation domain of AlgB was dispensable for alginate biosynthetic gene (i.e., algD operon) activation. However, it remained unclear whether AlgB stimulated algD transcription directly or indirectly. In this study, microarray analyses were used to examine a set of potential AlgB-dependent, KinB-independent genes in a PAO1 mucA background that overlapped with genes induced by d-cycloserine, which is known to activate algD expression. This set contained only the algD operon plus one other gene that was shown to be uninvolved in alginate production. This suggested that AlgB promotes alginate production by directly binding to the algD promoter (PalgD). Chromosome immunoprecipitation revealed that AlgB bound in vivo to PalgD but did not bind when AlgB had an R442E substitution that disrupted the DNA binding domain. AlgB also showed binding to PalgD fragments in an electrophoretic mobility shift assay at pH 4.5 but not at pH 8.0. A direct systematic evolution of ligands by exponential enrichment approach showed AlgB binding to a 50-bp fragment located at bp −224 to −274 relative to the start of PalgD transcription. Thus, AlgB belongs to a subclass of NtrC family proteins that can activate promoters which utilize a sigma factor other than σ54, in this case to stimulate transcription from the σ22-dependent PalgD promoter.

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