scholarly journals Apolipoprotein H Promoter Polymorphisms in Relation to Lupus and Lupus-related Phenotypes

2009 ◽  
Vol 36 (2) ◽  
pp. 315-322 ◽  
Author(s):  
SANGITA SURESH ◽  
F. YESIM K. DEMIRCI ◽  
ERIN JACOBS ◽  
AMY H. KAO ◽  
ELISA Y. RHEW ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Disease Risk ◽  
Gene Promoters ◽  
Nucleotide Polymorphisms ◽  
Promoter Polymorphisms ◽  
Mobility Shift ◽  
Association Analyses ◽  
Gene Assay ◽  
Mobility Shift Assay

Objective.Sequence variation in gene promoters is often associated with disease risk. We tested the hypothesis that common promoter variation in the APOH gene (encoding for ß2-glycoprotein I) is associated with systemic lupus erythematosus (SLE) risk and SLE-related clinical phenotypes in a Caucasian cohort.Methods.We used a case-control design and genotyped 345 women with SLE and 454 healthy control women for 8APOHpromoter single-nucleotide polymorphisms (SNP; –1284C>G, –1219G>A, –1190G>C, –759A>G, –700C>A, –643T>C, –38G>A, and –32C>A).Association analyses were performed on single SNP and haplotypes. Haplotype analyses were performed using EH (Estimate Haplotype–frequencies) and Haploview programs.In vitroreporter gene assay was performed in COS-1 cells. Electrophoretic mobility shift assay (EMSA) was performed using HepG2 nuclear cells.Results.Overall haplotype distribution of theAPOHpromoter SNP was significantly different between cases and controls (p = 0.009). The –643C allele was found to be protective against carotid plaque formation (adjusted OR 0.37, p = 0.013) among patients with SLE. The –643C allele was associated with a ~2-fold decrease in promoter activity as compared to wild-type –643T allele (mean ± standard deviation: 3.94 ± 0.05 vs 6.99 ± 0.68, p = 0.016). EMSA showed that the –643T>C SNP harbors a binding site for a nuclear factor. The –1219G>A SNP showed a significant association with the risk of lupus nephritis (age-adjusted OR 0.36, p = 0.016).Conclusion.Our data indicate thatAPOHpromoter variants may be involved in the etiology of SLE, especially the risk for autoimmune-mediated cardiovascular disease.

Cloning of rat and mouse aquaporin-2 gene promoters and identification of a negative cis-regulatory element

1997 ◽  
Vol 273 (2) ◽  
pp. F264-F273 ◽  
Author(s):  
T. Rai ◽  
S. Uchida ◽  
F. Marumo ◽  
S. Sasaki
Keyword(s):  
Specific Binding ◽  
Regulatory Element ◽  
Gene Promoter ◽  
Nuclear Extract ◽  
Gene Promoters ◽  
Specific Expression ◽  
Cis Element ◽  
Aquaporin 2 ◽  
Gene Assay

The promoters of rat and mouse aquaporin-2 (AQP-2) genes were cloned and compared with that of human genes. Nucleotide identity up to -593 bp was 62%, and consensus sequences such as TATA box and adenosine 3',5'-cyclic monophosphate responsive element were conserved. Deoxyribonuclease I footprint assay revealed a footprinted region at -210 to -184 bp in rat AQP-2 gene promoter produced by nuclear extract from nonexpressing (liver) tissue. The sequence of this region included a GATA motif but otherwise showed no homology with any other previously known cis-elements. Electromobility shift assay and ultraviolet cross-linking analysis confirmed that specific binding proteins to this element were present in kidney, spleen, and liver and that these proteins were distinct from GATA factors. Both deletion and mutation of this cis-element abolished the protein DNA binding and increased promoter activity in in vitro reporter gene assay using rat cultured hepatocyte Ac2F cells, suggesting the negative regulatory role of this cis-element. These results indicate that tissue-specific expression of AQP-2 gene may in part be regulated by this novel negative acting cis-element.

scholarly journals Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf

1995 ◽  
Vol 311 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M A Bevilacqua ◽  
M C Faniello ◽  
P D′Agostino ◽  
B Quaresima ◽  
M T Tiano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mobility Shift ◽  
Differentiated Cells ◽  
Binding Factor ◽  
H Gene ◽  
Ferritin Gene ◽  
Mobility Shift Assay ◽  
Promoter Interaction

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

2009 ◽  
Vol 425 (1) ◽  
pp. 235-243 ◽  
Author(s):  
Raquel  Castro-Prego ◽  
Mónica Lamas-Maceiras ◽  
Pilar Soengas ◽  
Isabel Carneiro ◽  
Isabel González-Siso ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxygen Availability ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Cross Links ◽  
Mobility Shift Assay ◽  
Transcriptional Adaptation

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1–Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides −557 to −376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

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 Transcriptional regulation of human and murine 17β-hydroxysteroid dehydrogenase type-7 confers its participation in cholesterol biosynthesis

2006 ◽  
Vol 37 (1) ◽  
pp. 185-197 ◽  
Author(s):  
Thomas Ohnesorg ◽  
Brigitte Keller ◽  
Martin Hrabé de Angelis ◽  
Jerzy Adamski
Keyword(s):  
Transcriptional Regulation ◽  
Binding Sites ◽  
Sequence Similarity ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Mobility Shift Assay

In both humans and mice, 17β-hydroxysteroid dehydrogenase type-7 (HSD17B7) was described as possessing dual enzymatic functionality. The enzyme was first shown to be able to convert estrone to estradiol in vitro. Later involvement of this enzyme in postsqualene cholesterol biosynthesis was postulated (conversion of zymosterone to zymosterol) and could be proven in vitro. In this work, we performed a detailed analysis of the transcriptional regulation of both the human and murine genes. Despite relatively low sequence similarity, both promoters contain similar contexts of transcription factor-binding sites. The participation of these sites in transcriptional regulation of HSD17B7 was proven by electro-mobility shift assay and site-directed mutagenesis of the corresponding binding sites. We describe novel involvement of vitamin D receptor/retinoid X receptor and provide new information on the regulation of HSD17B7 expression by sterol regulatory element-binding protein and hepatocyte nuclear factor 4, the latter known from other genes of cholesterogenic enzymes. The results of our study provide unequivocal evidence for a role of HSD17B7 in cholesterol biosynthesis.

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 In VitroAntiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Oi Kuan Choong ◽  
Parvaneh Mehrbod ◽  
Bimo Ario Tejo ◽  
Abdul Rahman Omar
Keyword(s):  
Viral Genome ◽  
Triple Helix ◽  
Antiviral Agents ◽  
Genome Copy ◽  
Mobility Shift ◽  
Feline Infectious Peritonitis ◽  
Copy Numbers ◽  
Mobility Shift Assay ◽  
Feline Coronavirus

Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. Thein vitroantiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10from 1014in the virus-inoculated cells to 109in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

Mechanical strain-induced proliferation and signaling in pulmonary epithelial H441 cells

2000 ◽  
Vol 279 (1) ◽  
pp. L43-L51 ◽  
Author(s):  
Patricia R. Chess ◽  
Liana Toia ◽  
Jacob N. Finkelstein
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Mechanical Strain ◽  
Mobility Shift ◽  
Pulmonary Epithelial Cells ◽  
Mitogen Activated Protein ◽  
Mobility Shift Assay

Pulmonary epithelial cells are exposed to mechanical strain during physiological breathing and mechanical ventilation. Strain regulates pulmonary growth and development and is implicated in volutrauma-induced fibrosis. The mechanisms of strain-induced effects are not well understood. It was hypothesized that mechanical strain induces proliferation of pulmonary epithelial cells and that this is mediated by signals initiated within seconds of strain. To test this hypothesis, human pulmonary adenocarcinoma H441 cells were strained in vitro. Cyclic as well as tonic strain resulted in increased cellular proliferation. Western blot analysis of strained cells demonstrated three newly phosphorylated tyrosine residues within 30 s of strain. Phosphorylation of mitogen-activated protein kinases p42/44 increased, electrophoretic mobility shift assay demonstrated activation of transcription factor activating protein-1, and immunohistochemistry demonstrated increased phosphorylation of c- jun in response to strain. The tyrosine kinase inhibitor genistein blocked the strain-induced proliferation. We conclude that strain induces proliferation in pulmonary epithelial cells and that tyrosine kinase activity is necessary to signal the proliferative response to mechanical strain.

DNA mobility shift assay as a tool for the detection of anti-dsDNA antibodies in sera from discoid lupus erythematosus patients

2012 ◽  
Vol 39 (7) ◽  
pp. 602-607 ◽  
Author(s):  
Jacqueline KEYHANI ◽  
Mashallah AHADI ◽  
Ezzatollah KEYHANI ◽  
Zahra NARAGHI ◽  
Safar SHAMOHAMMADI
Keyword(s):  
Lupus Erythematosus ◽  
Discoid Lupus Erythematosus ◽  
Mobility Shift ◽  
Dna Mobility ◽  
Mobility Shift Assay ◽  
Dsdna Antibodies
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