scholarly journals Subinhibitory Concentrations of Clinically-Relevant Antimicrobials Affect Resistance-Nodulation-Division Family Promoter Activity in Acinetobacter baumannii

2021 ◽  
Vol 12 ◽  
Author(s):  
Sonia Prieto Martin Gil ◽  
Ana Tajuelo ◽  
Mireia López-Siles ◽  
Michael J. McConnell
Keyword(s):  
Acinetobacter Baumannii ◽  
Growth Phase ◽  
Promoter Activity ◽  
Regulatory System ◽  
Efflux Pumps ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Reporter System ◽  
Resistance Nodulation Division ◽  
Subinhibitory Concentrations

Efflux pumps contribute to multidrug resistance in Acinetobacter baumannii due to their ability to expel a wide variety of structurally unrelated compounds. This study aimed to characterize the effect of subinhibitory concentrations of clinically-relevant antibiotics and disinfectants on the promoter activity of members of the Resistance-Nodulation-Division (RND) family in A. baumannii. The promoter regions from three RND efflux pumps (AdeABC, AdeFGH and AdeIJK) and the AdeRS regulatory system from three different A. baumannii strains (ATCC 17961, ATCC 17978, and ATCC 19606) were cloned into a luciferase reporter system (pLPV1Z). Promoter activity was quantitatively assessed in both exponential and stationary phase cultures after exposure to subinhibitory concentrations of four antibiotics from different classes (rifampicin, meropenem, tigecycline and colistin) and two disinfectants (ethanol and chlorhexidine). Subinhibitory concentrations of the compounds tested had variable effects on promoter activity that were highly dependent on the A. baumannii strain, the compound tested and the growth phase. Fold changes in AdeABC promoter activity ranged from 1.97 to 113.7, in AdeFGH from −5.6 to 1.13, in AdeIJK from −2.5 to 2, and in AdeRS from −36.2 to −1.32. Taken together, these results indicate that subinhibitory concentrations of clinically-relevant antibiotics and disinfectants affect the promoter activity of RND family members in A. baumannii in a strain and growth phase dependent manner. These results may have important implications for the treatment of infections caused by A. baumannii.

scholarly journals Synergistic Up-Regulation of Vascular Endothelial Growth Factor (VEGF) Expression in Macrophages by Adenosine A2AReceptor Agonists and Endotoxin Involves Transcriptional Regulation via the Hypoxia Response Element in the VEGF Promoter

2007 ◽  
Vol 18 (1) ◽  
pp. 14-23 ◽  
Author(s):  
Madhuri Ramanathan ◽  
Grace Pinhal-Enfield ◽  
Irene Hao ◽  
Samuel Joseph Leibovich
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Transcriptional Regulation ◽  
Growth Factor ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Vegf Expression ◽  
Endothelial Growth Factor

Macrophages are an important source of vascular endothelial growth factor (VEGF). Adenosine A2Areceptor (A2AR) agonists with Toll-like receptor (TLR) 2, 4, 7, and 9 agonists synergistically induce macrophage VEGF expression. We show here using VEGF promoter-luciferase reporter constructs that the TLR4 agonist Escherichia coli lipopolysaccharide (LPS) and the A2AR agonists NECA and CGS21680 synergistically augment VEGF transcription in macrophages and that the HRE in the VEGF promoter is essential for this transcription. We examined whether LPS and/or NECA induce HIF-1α expression. HIF-1α mRNA levels were increased in LPS-treated macrophages in an NF-κB–dependent manner; NECA strongly increased these levels in an A2AR-dependent manner. LPS induced luciferase expression from a HIF-1α promoter-luciferase construct in an A2AR-independent manner. Further stimulation with NECA did not increase HIF-1α promoter activity, indicating that the A2AR-dependent increase in HIF-1α mRNA is post-transcriptional. LPS/NECA treatment also increased HIF-1α protein and DNA binding levels. Deletion of putative NF-κB–binding sites from the VEGF promoter did not affect LPS/NECA-induced VEGF promoter activity, suggesting that NF-κB is not directly involved in VEGF transcription. Taken together, these data indicate that LPS/NECA-induced VEGF expression involves transcriptional regulation of the VEGF promoter by HIF-1α through the HRE. HIF-1α is transcriptionally induced by LPS and post-transcriptionally up-regulated in an A2AR-dependent manner.

Characterization of Hepcidin-Inducing Cytokines in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2001-2001
Author(s):  
Ken Maes ◽  
Alissa Huston ◽  
David Roodman ◽  
Seth Rivera ◽  
Elizabeta Nemeth ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Promoter Activity ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Advisory Committees ◽  
Hepcidin Expression ◽  
Equity Ownership

Abstract Abstract 2001 Poster Board I-1023 Introduction: Hepcidin, the principal iron-regulatory hormone, plays an important role in the development of anemia of inflammation and other iron-restricted anemias. Patients with multiple myeloma (MM) frequently present with anemia not attributable to a known mechanism. We previously found that hepcidin is increased in MM and thus could cause or contribute to the anemia of MM. The BMP and IL-6 pathways are the two known major transcriptional regulators of hepcidin. Methods: To identify cytokines that increase hepcidin in MM patients, we screened patient sera with an in vitro cellular reporter system, consisting of human hepatoma 7 cell line (HuH7) and the hepcidin promoter-firefly luciferase reporter. Using site-directed mutagenesis, the promoter was mutated at the STAT3-binding site (STAT3-BS) and/or two BMP responsive elements (BREs), sequences known to be involved in the regulation of hepcidin expression by IL-6 and BMPs, respectively. Results: As expected, recombinant IL-6 and BMP-4, -6 and -9 activated wild-type hepcidin promoter activity several fold. Of note, IL-6 and BMP-9 interacted synergistically at low doses, at the level of the promoter. Mutations in STAT3-BS abrogated the response to IL-6. Mutations in either BRE site by itself did not abolish the response to BMPs, but concurrent mutagenesis of both sites resulted in a complete loss of hepcidin response. Importantly, STAT3-BS and BREs affected hepcidin promoter response independently from each other. Using the in vitro system, we compared sera from six MM patients with previously measured serum hepcidin levels to sera from healthy controls. Sera of four patients with high hepcidin and one with low hepcidin significantly induced hepcidin promoter activity, while serum of another patient with low hepcidin did not. Mutations in STAT3-BS only abrogated the response to two patient sera, both of which had high hepcidin. Mutations in two BREs abrogated the response in all six sera as did the triple mutation involving STAT3-BS and both BREs. Conclusions: We could separately interrogate the signaling pathways by which IL-6 and BMPs induce hepcidin transcription, allowing us to discriminate between the effects of IL-6-like or BMP-like cytokines in each patient serum. BMP-like cytokines were involved in the upregulation of hepcidin in all tested MM patients, and in some patients IL-6 or related cytokines contributed as well, either independently or through a synergistic interaction. No residual activation of hepcidin promoter by other factors was noted. Antibody neutralization may identify the specific myeloma-associated cytokines that stimulate hepcidin production through the two canonical pathways. Disclosures: Roodman: Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Acceleron: Consultancy. Nemeth:Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Xenon Pharmaceuticals: Consultancy, Equity Ownership.

scholarly journals Gastrin regulates the TFF2 promoter through gastrin-responsive cis-acting elements and multiple signaling pathways

2007 ◽  
Vol 292 (6) ◽  
pp. G1726-G1737 ◽  
Author(s):  
Shuiping Tu ◽  
Alfred L. Chi ◽  
SeonHee Lim ◽  
Guanglin Cui ◽  
Zina Dubeykovskaya ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Growth Factor Receptor ◽  
Gastric Cancer Cell Line ◽  
Cancer Cell Line ◽  
Gastric Fundus ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Cis Acting

Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCKB receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.

scholarly journals Differential regulation of the two-component regulatory system senX3-regX3 in Mycobacterium tuberculosis

Microbiology ◽  
2014 ◽  
Vol 160 (6) ◽  
pp. 1125-1133 ◽  
Author(s):  
Dalin Rifat ◽  
Petros C. Karakousis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Promoter Activity ◽  
Regulatory System ◽  
Differential Regulation ◽  
Northern Analysis ◽  
Pcr Analysis ◽  
Reporter System ◽  
Two Component ◽  
Phosphate Depletion

The highly successful pathogen Mycobacterium tuberculosis (Mtb) has evolved strategies to adapt to various stress conditions, thus promoting survival within the infected host. The two-component regulatory system (2CRS) senX3-regX3, which has been implicated in the Mtb response to inorganic phosphate depletion, is believed to behave as an auto-regulatory bicistronic operon. Unlike other 2CRS, Mtb senX3-regX3 features an intergenic region (IR) containing several mycobacterium interspersed repetitive units (MIRU) of unknown function. In this study, we used a lacZ reporter system to study the promoter activity of the 5′ untranslated region of senX3, and that of various numbers of MIRUs in the senX3-regX3 IR, during axenic Mtb growth in nutrient-rich broth, and upon exposure to growth-restricting conditions. Activity of the senX3 promoter was induced during phosphate depletion and nutrient starvation, and IR promoter activity under these conditions was directly proportional to the number of MIRUs present. Quantitative reverse transcriptase (qRT)-PCR analysis of exponentially growing Mtb revealed monocistronic transcription of senX3 and regX3, and, to a lesser degree, bicistronic transcription of the operon. In addition, we observed primarily monocistronic upregulation of regX3 during phosphate depletion of Mtb, which was confirmed by Northern analysis in wild-type Mtb and by RT-PCR in a senX3-disrupted mutant, while upregulation of regX3 in nutrient-starved Mtb was chiefly bicistronic. Our findings of differential regulation of senX3-regX3 highlight the potential regulatory role of MIRUs in the Mtb genome and provide insight into the regulatory mechanisms underlying Mtb adaptation to physiologically relevant conditions.

scholarly journals Physiological Analysis of the Expression of the Styrene Degradation Gene Cluster in Pseudomonas fluorescensST

2000 ◽  
Vol 66 (4) ◽  
pp. 1305-1310 ◽  
Author(s):  
Pedro Miguel Santos ◽  
Janet Martha Blatny ◽  
Ilaria Di Bartolo ◽  
Svein Valla ◽  
Elisabetta Zennaro
Keyword(s):  
Growth Phase ◽  
Exponential Growth ◽  
Carbon Sources ◽  
Lactate Concentration ◽  
Regulatory System ◽  
Exponential Growth Phase ◽  
Galactosidase Activity ◽  
Reporter System ◽  
Catabolic Genes ◽  
Physiological Analysis

ABSTRACT The effects of different carbon sources on expression of the styrene catabolism genes in Pseudomonas fluorescens ST were analyzed by using a promoter probe vector, pPR9TT, which contains transcription terminators upstream and downstream of the β-galactosidase reporter system. Expression of the promoter of thestySR operon, which codes for the styrene two-component regulatory system, was found to be constitutive and not subject to catabolite repression. This was confirmed by the results of an analysis of the stySR transcript in P. fluorescensST cells grown on different carbon sources. The promoter of the operon of the upper pathway, designated PstyA, was induced by styrene and repressed to different extents by organic acids or carbohydrates. In particular, cells grown on succinate or lactate in the presence of styrene started to exhibit β-galactosidase activity during the mid-exponential growth phase, before the preferred carbon sources were depleted, indicating that there is a threshold succinate and lactate concentration which allows induction of styrene catabolic genes. In contrast, cells grown on glucose, acetate, or glutamate and styrene exhibited a diauxic growth curve, and β-galactosidase activity was detected only after the end of the exponential growth phase. In each experiment the reliability of the reporter system constructed was verified by comparing the β-galactosidase activity and the activity of the styrene monooxygenase encoded by the first gene of the styrene catabolic operon.

scholarly journals Nuclear receptors RXRα:RARα are repressors for human MRP3 expression

2007 ◽  
Vol 292 (5) ◽  
pp. G1221-G1227 ◽  
Author(s):  
Wensheng Chen ◽  
Shi-Ying Cai ◽  
Shuhua Xu ◽  
Lee A. Denson ◽  
Carol J. Soroka ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Site Mutation ◽  
Transcription Factor Sp1 ◽  
Gc Box ◽  
Rna Knockdown ◽  
Interfering Rna

Multidrug resistance-associated protein MRP3/Mrp3 (ABCC3) is upregulated in cholestasis, an adaptive response that may protect the liver from accumulation of toxic compounds, such as bile salts and bilirubin conjugates. However, the mechanism of this upregulation is poorly understood. We and others have previously reported that fetoprotein transcription factor/liver receptor homolog-1 is an activator of MRP3/Mrp3 expression. In searching for additional regulatory elements in the human MRP3 promoter, we have now identified nuclear receptor retinoic X receptor-α:retinoic acid receptor-α (RXRα:RARα) as a repressor of MRP3 activation by transcription factor Sp1. A luciferase reporter assay demonstrated that cotransfection of transcription factor Sp1 stimulates the MRP3 promoter activity and that additions of RXRα:RARα abrogated this activation in a dose-dependent manner. Site mutations and gel shift assays have identified a Sp1 binding GC box motif at −113 to −108 nts upstream from the MRP3 translation start site, where RXRα:RARα specifically reduced Sp1 binding to this site. Mutation of the GC box also reduced MRP3 promoter activity. The functional role of RXRα:RARα as a repressor of MRP3 expression was further confirmed by RARα small-interfering RNA knockdown in HepG2 cells, which upregulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXRα:RARα act in concert to regulate MRP3 expression. Since RXRα:RARα expression is diminished by cholestatic liver injury, loss of RXRα:RARα may lead to upregulation of MRP3/Mrp3 expression in these disorders.

Biofilm Formation Restrained by Subinhibitory Concentrations of Tigecyclin in Acinetobacter baumannii Is Associated with Downregulation of Efflux Pumps

Chemotherapy ◽  
2016 ◽  
Vol 62 (2) ◽  
pp. 128-133 ◽  
Author(s):  
Huale Chen ◽  
Jianming Cao ◽  
Cui Zhou ◽  
Haiyang Liu ◽  
Xiaoxiao Zhang ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Real Time ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Subinhibitory Concentrations

Background: Tigecycline, one of the few therapeutic options against multidrug-resistant Acinetobacter baumannii, reaches subinhibitory serum concentrations only with cautious clinical dosing and pharmacokinetics. Subinhibitory concentrations of tigecycline might induce an A. baumannii biofilm. Methods: Biofilm formation was assessed via the crystal violet staining method. We further analyzed the main biofilm components with NaIO4, proteinase K, and DNase. Real-time RT-PCR was applied for quantitative detection of biofilm potential-associated genes. Results: In this study, A. baumannii proved to be a strong biofilm producer, and we found that proteins and extracellular DNA are crucial components of the A. baumannii biofilm. Quantitative real-time RT-PCR revealed positive correlations between biofilm formation restrained by subinhibitory concentrations of tigecycline and the expression of biofilm potential-associated genes, especially the AdeFGH efflux pump gene. Conclusion: Our results suggest that downregulation of efflux pumps, especially the AdeFGH efflux pump, is probably responsible for the decline in biofilm formation in A. baumannii treated with subinhibitory concentrations of tigecyclin.

scholarly journals Quantitative Assessment of Pdx1 Promoter Activity in Vivo Using a Secreted Luciferase Reporter System

Endocrinology ◽  
2013 ◽  
Vol 154 (11) ◽  
pp. 4388-4395 ◽  
Author(s):  
Wataru Nishimura ◽  
Koki Eto ◽  
Atsushi Miki ◽  
Motohito Goto ◽  
Miho Kawaguchi ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Quantitative Assessment ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Luciferase Reporter System

PPARγ regulates fabp4 expression to increase DHA content in golden pompano (Trachinotus ovatus) hepatocytes

2021 ◽  
pp. 1-9
Author(s):  
Caixia Lei ◽  
Bin Fan ◽  
Jingjing Tian ◽  
Mengmeng Li ◽  
Yuanyou Li
Keyword(s):  
Promoter Activity ◽  
Core Promoter ◽  
Promoter Sequence ◽  
Active Role ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Pufa Content ◽  
Farmed Fish ◽  
Fatty Acid Binding ◽  
Trachinotus Ovatus

Abstract N-3 long-chain (≥C20) PUFA (LC-PUFA) are vital fatty acids for fish and humans. As a main source of n-3 LC-PUFA for human consumers, the n-3 LC-PUFA content of farmed fish is important. Previously, we identified fatty acid-binding protein (fabp)-4 as a candidate gene for regulating the n-3 LC-PUFA content. Herein, we further assessed the role of fabp4 in this process. First, a 2059 bp promoter sequence of fabp4 in Trachinotus ovatus was cloned and, using progressive deletion, determined −2006 bp to −1521 bp to be the core promoter sequence. The PPAR-γ binding sites were predicted to occur in this region. A luciferase reporter assay showed that the promoter activity of fabp4 decreased following mutation of the PPARγ binding site and that PPARγ increased the fabp4 promoter activity in a dose-dependent manner, implying that T. ovatus fabp4 is a target of PPARγ. The overexpression of fabp4 or PPARγ increased the DHA content in hepatocytes, whereas suppression of their expression diminished this effect, suggesting that both fabp4 and PPARγ play an active role in regulating DHA content. Moreover, the inhibition of fabp4 attenuated the increase in PPARγ-mediated DHA content, and the overexpression of fabp4 alleviated this effect. Collectively, our findings indicated that fabp4, which is controlled by PPARγ, plays an important role in DHA content regulation. The new regulation axis can be considered a promising novel target for increasing the n-3 LC-PUFA content in T. ovatus.

scholarly journals Saccharomyces cerevisiae JEN1 Promoter Activity Is Inversely Related to Concentration of Repressing Sugar

2004 ◽  
Vol 70 (1) ◽  
pp. 8-17 ◽  
Author(s):  
Prima Chambers ◽  
Aminatu Issaka ◽  
Sean P. Palecek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Concentration ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Regulatory Elements ◽  
Dependent Manner ◽  
Diauxic Shift ◽  
Reporter System ◽  
Genomic Expression

ABSTRACT When carbon sources are changed, Saccharomyces cerevisiae transcriptional patterns drastically change. To identify genes whose transcription can be used to quantitatively measure sugar concentrations, we searched genomic expression databases for a set of genes that are highly induced during the diauxic shift, and we used the promoters from these genes to drive expression of green fluorescent protein (GFP). Certain sugars, including glucose, fructose, and mannose, repress the promoter of JEN1, which encodes a lactate-pyruvate transporter, in a dose-dependent manner. Nonrepressing carbon sources include galactose, raffinose, ethanol, lactate, and glycerol. JEN1 promoter activity is a linear function of glucose concentration when organisms are grown at a steady-state glucose concentration below 1 g/liter. JEN1 promoter repression is specific to carbon source; heat or cold shock, osmotic stress, DNA damage, and nitrogen starvation do not significantly affect promoter activity. Activation of the JEN1 promoter requires the Snf1 protein kinase, but multiple regulatory elements most likely combine to provide the linear relationship between JEN1 promoter activity and sugar concentration. Thus, a JEN1 promoter-reporter system appears to provide a good living cell biosensor for the concentration of certain sugars. The JEN1 promoter also permits quantitative regulation of cellular functions not normally controlled by sugar concentrations. For example, a strain expressing FLO1 under control of the JEN1 promoter flocculates at a low glucose concentration.

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