DNA Mobility Shift Assays Research Articles

Insights into the Biological Properties of Ligands and Identity of Operator Site for LanK Protein Involved in Landomycin Production.

LanK is a TetR type regulatory protein that coordinates the late steps of the biosynthesis of the landomycin family of antitumor angucyclic polyketides and their export from the cells of Streptomyces cyanogenus S136. We recently described the structure of LanK and showed that it is the carbohydrate portion of the landomycins that is responsible for abrogating the repressing effect of LanK on landomycin production and export. The effect has been established in a series of in vitro tests using synthetic analogs of the landomycin carbohydrate chains. Whether such synthetic compounds would function as effector molecules for LanK under in vivo conditions remained unknown. Furthermore, the location and identity of LanK operator sites within the lanK-lanJ intergenic region (lanKJp) was unknown. Here we report that methoxyphenyl analogs of tri- and hexasaccharide chains of landomycins cannot function as LanK ligands when applied externally to the reporter strain. The lability of these compounds to cellular media and/or their poor penetration into the cells could explain our observations. The LanK operator site has been mapped to a 14-bp region of lanKJp that includes a plausible -35 site upstream of the lanK start codon in a series of electrophoretic DNA mobility shift assays. This opens the door to studies of the DNA-LanK interaction at a single nucleotide resolution level.

DNA interaction and BSA binding of O-vanillin-based new Schiff base Co(III) and Ni(II) complexes: Theoretical, experimental, antibacterial and anticancer studies

In the present work, two new mononuclear CoIII and NiII complexes containing H2valmea Schiff base ligand (H2valmea = 2-(((2-hydroxyethyl)imino)methyl)-6-methoxyphenol) were synthesized. X-ray crystallography, besides other techniques such as CHN, FT-IR and UV–Vis spectroscopy, determined the exact structures of both complexes. Bio-interactions (BSA and DNA binding), the antineoplastic ability and basal cell toxicity of complexes were investigated by various experimental and docking methods. Fluorescence emission and UV–Vis absorbance experiments confirmed the static mechanism in the interaction of both complexes with protein. Site marker competitive and tryptophan quenching experiments alongside docking studies revealed that CoIII complex preferred site I while NiII complex preferred site II of BSA. The main type of the complexes-protein interaction was hydrophobic forces, and FRET theory indicated that transferring energy was feasible for mononuclear complexes. Investigation of the complex-DNA interactions were done by absorption spectroscopy, ethidium bromide (EtBr) fluorescence displacement experiment, docking studies, cyclic voltammetry and viscosity measurements. All these studies suggested that NiII complex interacted with DNA by mainly intercalation mode of binding. In contrast, CoIII complex was a significantly weaker intercalator because it could not compete with EtBr in binding with DNA and revealed a lower MolDock score for intercalation than ethidium bromide in docking studies. These observations are confirmed by in vitro DNA mobility shift assay that indicated the strong inter-strand intercalation affinity of the NiII complex. Also, using the flow cytometry method, the apoptosis/necrosis pathway of the NiII complex was studied against A549 cells. The antibacterial assay showed the great bacteriocidal potency of NiII complex, while in the case of E.coli treatment, a moderate bacteriostatic behavior is seen.

Pyronaridine exerts potent cytotoxicity on human breast and hematological cancer cells through induction of apoptosis.

The potent antimalarial drug pyronaridine (PND) was tested for its potential as an anticancer drug. After exposing cancerous (17) and non-cancerous (2) cells to PND for 72 hr, PND was found to exhibit consistent and potent cytotoxic activity at low micromolar (μM) concentrations that ranged from 1.6 μM to 9.4 μM. Moreover, PND exerted a significant selective cytotoxicity index (SCI) on five out of seven breast cancer cell lines tested, with favorable values of 2.5 to 4.4, as compared with the non-cancerous breast MCF-10A cell line. By using the same comparison, PND exhibited a significant SCI on three out of four leukemia/lymphoma cell lines with promising values of 3.3 to 3.5. One breast cancer and one leukemia cell line were tested further in order to determine the likely mode of action of PND. PND was found to consistently elicit phosphatidylserine externalization, mitochondrial depolarization, and DNA fragmentation, in both the triple negative MDA-MB-231 breast cancer and HL-60 leukemia cell lines. In addition, PND treatment altered cell cycle progression in both cancer cells. Subsequent DNA mobility-shift assays, UV-Visible spectroscopic titrations, and circular dichroism (CD) experiments revealed that PND intercalates with DNA. The findings presented in this study indicates that PND induces apoptosis and interfered with cell cycle progression of cancer cell lines and these results indicate that this drug has the potential as a repurposed drug for cancer therapy.

The extracellular matrix protein mindin attenuates colon cancer progression by blocking angiogenesis via Egr-1-mediated regulation.

Mindin, a secreted, highly conserved extracellular matrix (ECM) protein, exerts a broad spectrum of effects on the innate immune system. However, its function in colorectal cancer (CRC) progression is not well established, and its upstream regulation mechanisms remain unclear. Contrary to previous reports, this study used two different enzyme-linked immunosorbent assay (ELISA) kits to show that the serum level of mindin was significantly decreased in CRC patients and that this decreased level is more significantly associated with the early stages of the disease. To explore the regulation of mindin, we used a bioinformatics approach to predict potential transcription factors and determined that early growth response factor (Egr)-1 directly regulates mindin expression at the transcriptional level using dual luciferase, chromatin immunoprecipitation (ChIP) DNA and electrophoretic mobility shift assay (EMSA) methods. Egr-1 regulates mindin mRNA and protein expression in CRC cells, and the protein expression of both Egr-1 and mindin was significantly decreased in tumor lesions of patients compared with adjacent control tissues. Mindin is essential for Egr-1-mediated inhibition of endothelial cell tube formation, and mindin inhibits endotheliocyte proliferation, migration and angiogenic sprouts in vitro. Overexpression of mindin suppressed xenograft tumor growth by blocking angiogenesis instead of directly suppressing CRC cell proliferation. Mechanically, mindin inhibits the hypoxia-induced HIF-1a and VEGFA protein expression in CRC cells and the phosphorylation of VEGFR-2 in endothelial cells. The results suggest that the serum level of mindin can be used as a novel biomarker for early detection of CRC and that the Egr-1/mindin axis is a potential therapeutic target for the inhibition of angiogenesis in CRC development.

H-NST Induces LEE Expression and the Formation of Attaching and Effacing Lesions in Enterohemorrhagic Escherichia coli

BackgroundEnteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli are important causes of morbidity and mortality worldwide. These enteric pathogens contain a type III secretion system (T3SS) responsible for the attaching and effacing (A/E) lesion phenotype. The T3SS is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. The H-NS-mediated repression of LEE expression is counteracted by Ler, the major activator of virulence gene expression in A/E pathogens. A regulator present in EPEC, H-NST, positively affects expression of H-NS regulon members in E. coli K-12, although the effect of H-NST on LEE expression and virulence of A/E pathogens has yet-to-be determined.ResultsWe examine the effect of H-NST on LEE expression and A/E lesion formation on intestinal epithelial cells. We find that H-NST positively affects the levels of LEE-encoded proteins independently of ler and induces A/E lesion formation. We demonstrate H-NST binding to regulatory regions of LEE1 and LEE3, the first report of DNA-binding by H-NST. We characterize H-NST mutants substituted at conserved residues including Ala16 and residues Arg60 and Arg63, which are part of a potential DNA-binding domain. The single mutants A16V, A16L, R60Q and the double mutant R60Q/R63Q exhibit a decreased effect on LEE expression and A/E lesion formation. DNA mobility shift assays reveal that these residues are important for H-NST to bind regulatory LEE DNA targets. H-NST positively affects Ler binding to LEE DNA in the presence of H-NS, and thereby potentially helps Ler displace H-NS bound to DNA.ConclusionsH-NST induces LEE expression and A/E lesion formation likely by counteracting H-NS-mediated repression. We demonstrate that H-NST binds to DNA and identify arginine residues that are functionally important for DNA-binding. Our study suggests that H-NST provides an additional means for A/E pathogens to alleviate repression of virulence gene expression by H-NS to promote virulence capabilities.

Novel function of the poly(c)-binding protein α-CP2 as a transcriptional activator that binds to single-stranded DNA sequences

α-complex protein 2 (α-CP2) is known as an RNA-binding protein that interacts in a sequence-specific manner with single-stranded polycytosine [poly(C)]. This protein is involved in various post-transcriptional regulations, such as mRNA stabilization and translational regulation. In this study, the full-length mouse α-CP2 gene was expressed in an insoluble form with an N-terminal histidine tag in Escherichia coli and purified for homogeneity using affinity column chromatography. Its identity was confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Recombinant α-CP2 was expressed and refolded. The protein folding conditions for denatured α-CP2 were optimized. DNA and RNA electrophoretic mobility shift assays demonstrated that the recombinant α-CP2 is capable of binding to both single-stranded DNA and RNA poly(C) sequences. Furthermore, plasmids expressing α-CP2 activated the expression of a luciferase reporter when co-transfected with a single-stranded (pGL-SS) construct containing a poly(C) sequence. To our knowledge, this study demonstrates for the first time that α-CP2 functions as a transcriptional activator by binding to a single-stranded poly(C) sequence.

Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15.

The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15–20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn2+-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.

Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.

Transcriptional Regulation of the Group IIA Secretory Phospholipase A2 Gene by C/EBPδ in Rat liver and its Relationship to Hepatic Gluconeogenesis during Sepsis.

The present study was undertaken to test hypothesis that altered transcription of secretory Phospholipase A2 (sPLA2) gene in rat liver is regulated by CCAAT/enhancer binding protein δ (C/EBPδ), and to assess its relationship to hepatic gluconeogenesis during the progression of sepsis. Sepsis was induced by Cecal Ligation and Puncture (CLP). Experiments were divided into three groups, control, early sepsis (9 h after CLP), and late sepsis (18 h after CLP). DNA mobility and super shift assays reveal that C/EBP complexes in the liver consisted of at least three isoforms: C/EBPα, C/EBPβ, and C/EBPδ; and various C/EBP isoforms were capable of interacting with each other. Hepatocyte transfection experiments demonstrate that under normal conditions, binding of C/EBPδ to sPLA2 gene enhanced sPLA2 promoter activity and the binding resulted in an increase in hepatic gluconeogenesis. Under pathological conditions such as sepsis, binding of C/EBPδ to sPLA2 promoter increased during early and late phases of sepsis, and the increases in C/EBPδ binding correlated with increases in sPLA2 mRNA abundance and sPLA2 protein levels. Under otherwise the identical experimental conditions, hepatic gluconeogenesis was reduced during early and late phases of sepsis and the sepsis-induced reductions in liver gluconeogenesis were aggravated by binding of C/EBPδ to sPLA2 gene. These results link C/EBPδ binding to altered sPLA2 promoter, and to hepatic gluconeogenesis under normal and pathological conditions. It is suggested that C/EBPδ-sPLA2- hepatic gluconeogenesis may function as a signalling axis affecting glucose homeostasis during the progression of sepsis.

Modulation of cell wall synthesis and susceptibility to vancomycin by the two-component system AirSR in Staphylococcus aureus NCTC8325

BackgroundVancomycin has been the medication of last resort to cure infections caused by Staphylococcus aureus since the increase in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA). Some strains have developed vancomycin-intermediate resistance, which is generally associated with altered expression of or mutations in some part of the two-component system (TCS), such as GraSR, VraSR, and WalKR.ResultsWe deleted the AirSR TCS in S. aureus NCTC8325 and compared the resultant transcript levels with those of its parent strain using microarray analysis. The results indicated that more than 20 genes that are related to cell wall metabolism were down-regulated in the airSR mutant. The airSR mutant exhibited reduced autolysis rates and reduced viability in the presence of vancomycin. Real-time reverse transcription PCR and DNA mobility shift assays verified that AirR can directly bind to and regulate genes that function in cell wall metabolism (cap, pbp1, and ddl) and autolysis (lytM).ConclusionsAirSR acts as a positive regulator in cell wall biosynthesis and turnover in Staphylococcus aureus NCTC8325.

Crystal Structures of Two Transcriptional Regulators from Bacillus cereus Define the Conserved Structural Features of a PadR Subfamily

PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206–BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206–BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.

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

Discoid lupus erythematosus (DLE) is a form of local inflammatory autoimmune disease limited to the skin, involving essentially the face, scalp and ear. DLE occurs in genetically predisposed individuals, sunlight being an identified trigger. Diagnosis is made by clinical examination and histopathology; laboratory tests occasionally performed include anti-nuclear antibodies titers and presence of circulating antibodies against dsDNA. DLE patients have about a 10% chance of developing systemic lupus erythematosus (SLE), a systemic inflammatory autoimmune disease affecting a range of internal organs. Although elevated titers of anti-dsDNA antibodies is an earmark for lupus disease, they are detected in only 20-55% of DLE patients by routine laboratory tests such as enzyme-linked immunosorbent assay (ELISA). In this research, we applied an electrophoretic mobility shift assay (EMSA) in parallel with an ELISA for the detection of circulating anti-dsDNA in DLE patients. The assays were conducted on sera as well as on the immunoglobulin G fraction from sera of 24 DLE patients and of 24 healthy individuals. The EMSA was positive for all DLE patients while the ELISA was positive for only 36% of them; both assays were negative for the healthy individuals. EMSA conducted on the sera of 15 patients with lichen planus was negative in all cases. Results suggest that the EMSA is more sensitive than the routine tests used for the detection of anti-dsDNA in DLE, thus helping to improve early detection of the disease and, by extension, to better evaluate the factors triggering the disease.

Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain ofRhodobacter capsulatusMopB

The LysR-type regulator MopB represses transcription of several target genes (including the nitrogen-fixation gene anfA) in Rhodobacter capsulatus at high molybdenum concentrations. In this study, the isolated DNA-binding domain of MopB (MopBHTH) was overexpressed in Escherichia coli. Purified MopBHTH bound the anfA promoter as shown by DNA mobility-shift assays, demonstrating the function of the isolated regulator domain. MopBHTH was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.2 M lithium sulfate, 0.1 M phosphate/citrate pH 4.2, 20%(w/v) PEG 1000 at 291 K. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a=b=61.84, c=139.64 Å, α=β=90, γ=120°, and diffracted to 3.3 Å resolution at a synchrotron source.

A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis

Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d,eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in ~0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 fmol of total binding sequences were needed for an 11-point titration curve.

N-Glycosylation Gene DPAGT1 Is a Target of the Wnt/β-Catenin Signaling Pathway

Protein N-glycosylation and the Wnt/β-catenin signaling pathways play critical roles in development and cancer. Although N-glycosylation has been shown to influence Wnt signaling through its effects on Wnt ligands, it is unclear whether the Wnt/β-catenin pathway impacts protein N-glycosylation. In this study, we show that promoters of the first N-glycosylation gene, DPAGT1, from Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human epidermoid carcinoma (A253) cells contain the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus sequence. Treatment of cells with a Wnt activator, lithium chloride, up-regulated DPAGT1 transcript levels that correlated with an increase in the β-catenin abundance. Furthermore, exposure of cells to a Wnt receptor ligand, Wnt3a, resulted in an increase in the DPAGT1 transcript levels that was abrogated by the Wnt inhibitor, Dickkopf-1. DNA mobility shift assays revealed specific protein complexes at the DPAGT1 TCF/LEF binding region that were competed off with antibodies to either Tcf3/4 or β-catenin. Chromatin immunoprecipitation analysis confirmed the presence of β-catenin at the DPAGT1 promoter in vivo. In addition, the DPAGT1 TCF/LEF sequence drove the expression of the luciferase reporter gene. Furthermore, up-regulation of DPAGT1 transcripts by Wnt3a led to altered N-glycosylation of E-cadherin. Interestingly, the DPAGT1 TCF/LEF sequence also interacted with γ-catenin, a close homologue of β-catenin, although not in a lithium chloride-dependent manner. Our results provide the first evidence that the Wnt/β-catenin signaling pathway regulates the metabolic pathway of protein N-glycosylation by targeting DPAGT1 expression. Moreover, they suggest the existence of another regulatory mechanism involving the interaction of Tcf with γ-catenin at the DPAGT1 promoter.

Maternal obesity induces fibrosis in fetal myocardium of sheep

Maternal obesity (MO) has harmful effects on both fetal development and subsequent offspring health. The impact of MO on fetal myocardium development has received little attention. Fibrogenesis is regulated by the transforming growth factor-β (TGF-β)/p38 signaling pathway. Using the well-established model of MO in pregnant sheep, we evaluated the effect of MO on TGF-β/p38 and collagen accumulation in fetal myocardium. Nonpregnant ewes were assigned to a control diet [Con, fed 100% of National Research Council (NRC) nutrient recommendations] or obesogenic diet (OB, fed 150% of NRC recommendations) from 60 days before conception. Fetal ventricular muscle was sampled at 75 and 135 days of gestation (dG). At 75 dG, the expression of precursor TGF-β was 39.9 ± 9.9% higher (P < 0.05) in OB than Con fetal myocardium, consistent with the higher content of phosphorylated Smad3 in OB myocardium. The phosphorylation of p38 tended to be higher in OB myocardium (P = 0.08). In addition, enhanced Smad complexes were bound to Smad-binding elements in 75 dG OB fetal myocardium measured by DNA mobility shift assay (130.2 ± 26.0% higher, P < 0.05). Similar elevation of TGF-β signaling was observed in OB fetal myocardium at 135 dG. Total collagen concentration in OB was greater than Con fetal myocardium (2.42 ± 0.16 vs. 1.87 ± 0.04%, P < 0.05). Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 were higher in the Con group compared with OB sheep (43.86 ± 16.01 and 37.23 ± 7.97% respectively, P < 0.05). In summary, MO results in greater fetal heart connective tissue accumulation associated with an upregulated TGF-β/p38 signaling pathway at late gestation; such changes would be expected to negatively impact offspring heart function.

Relevance of individual Mo-box nucleotides to DNA binding by the related molybdenum-responsive regulators MopA and MopB in Rhodobacter capsulatus

Either of two related molybdenum-responsive regulators, MopA and MopB, of Rhodobacter capsulatus is sufficient to repress the nitrogen-fixation gene anfA. In contrast, MopA (but not MopB) activates mop, which codes for a molybdate (Mo)-binding molbindin. Both regulators bind to conserved cis-regulatory elements called Mo-boxes. Single-base substitution of two highly conserved nucleotides within the anfA-Mo-box (T21C and C24T) had little effect on regulator binding and anfA expression as shown by DNA mobility shift assays and reporter gene fusions, respectively. In contrast to C24T, mutation C24A strongly diminished binding and repression by MopA and MopB, showing that different nucleotide substitutions at the same position may have very different effects. A triple mutation destroying the left half-site of the mop-Mo-box completely abolished mop expression by MopA, demonstrating the importance of the mop-Mo-box for mop activation. Two point mutations (T23A and T24C) still allowed binding by MopA, but abolished mop activation, most likely because these nucleotides overlap with the RNA polymerase-binding site. A mutant mop promoter, in which the mop-Mo-box was exchanged against the anfA-Mo-box, allowed activation by MopA, showing that a former repressor-binding site may act as an activator-binding site depending on its location relative to the other promoter elements.

Synthesis, Crystal Structures, and DNA Binding Properties of Zinc(II) Complexes with 3‐Pyridine Aldoxime

The employment of 3-pyridine aldoxime, (3-py)CHNOH, in ZnII chemistry has afforded two novel compounds: [Zn(acac)2{(3-py)CHNOH}]·H2O (1·H2O) [where acac− is the pentane-2,4-dionato(-1) ion] and [Zn2(O2CMe)4{(3-py)CHNOH}2] (2). Complex 1·H2O crystallizes in the monoclinic space group P21/n. The ZnII ion is five-coordinated, surrounded by four oxygen atoms of two acac− moieties and by the pyridyl nitrogen atom of the (3-py)CHNOH ligand. Molecules of 1 interact with the water lattice molecules forming a 2D hydrogen-bonding network. Complex 2 crystallizes in the triclinic P-1 space group and displays a dinuclear paddle-wheel structure. Each ZnII exhibits a perfect square pyramidal geometry, with four carboxylate oxygen atoms at the basal plane and the pyridyl nitrogen of one monodentate (3-py)CHNOH ligand at the apex. DNA mobility shift assays were performed for the determination of the in vitro effect of both complexes on the integrity and the electrophoretic mobility of pDNA.

Positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis

OmpR has been demonstrated to negatively regulate the expression of the flagellar master operon flhDC in a wide variety of bacterial species. Here we report the positive regulation of flhDC expression by OmpR in Yersinia pseudotuberculosis. A sigma(70)-dependent promoter was identified by primer extension analysis and an active region with two conserved OmpR-binding sites around the flhDC promoter was confirmed. To confirm the regulation of flhDC expression by OmpR, flhDC as well as the downstream flagellar genes fliA, flgD, flgA, flgM, fliC and flaA were fused to lacZ, and decreased expression of all these genes in an ompR mutant (Delta ompR) was detected. Furthermore, Delta ompR was defective in bacterial motility and flagella synthesis. This defect was due to the low level of expression of flhDC in Delta ompR since overproduction of FlhDC in Delta ompR restored bacterial motility. The importance of two conserved OmpR-binding sites around the flhDC promoter region in the regulation of flhDC expression by OmpR was demonstrated by the fact that mutation of either one or both sites significantly decreased the promoter activity in the wild-type but not in Delta ompR. The binding of OmpR to these two sites was also demonstrated by DNA mobility shift assay. The possible mechanism underlying this positive regulation in Y. pseudotuberculosis is discussed. To our knowledge, this is the first report to demonstrate that OmpR positively regulates flhDC expression.

Functional and genetic analysis in type 2 diabetes of Liver X receptor alleles – a cohort study

BackgroundLiver X receptor alpha (LXRA) and beta (LXRB) regulate glucose and lipid homeostasis in model systems but their importance in human physiology is poorly understood. This project aimed to determine whether common genetic variations in LXRA and LXRB associate with type 2 diabetes (T2D) and quantitative measures of glucose homeostasis, and, if so, reveal the underlying mechanisms.MethodsEight common single nucleotide polymorphisms in LXRA and LXRB were analyzed for association with T2D in one French cohort (N = 988 cases and 941 controls), and for association with quantitative measures reflecting glucose homeostasis in two non-diabetic population-based samples comprising N = 697 and N = 1344 adults. Investigated quantitative phenotypes included fasting plasma glucose, serum insulin, and HOMAIR as measure of overall insulin resistance. An oral glucose tolerance test was performed in N = 1344 of adults. The two alleles of the proximal LXRB promoter, differing only at the SNP rs17373080, were cloned into reporter vectors and transiently transfected, whereupon allele-specific luciferase activity was measured. rs17373080 overlapped, according to in silico analysis, with a binding site for Nuclear factor 1 (NF1). Promoter alleles were tested for interaction with NF1 using direct DNA binding and transactivation assays.ResultsGenotypes at two LXRB promoter SNPs, rs35463555 and rs17373080, associated nominally with T2D (P values 0.047 and 0.026). No LXRA or LXRB SNP associated with quantitative measures reflecting glucose homeostasis. The rs17373080 C allele displayed higher basal transcription activity (P value < 0.05). The DNA-mobility shift assay indicated that oligonucleotides corresponding to either rs17373080 allele bound NF1 transcription factors in whole cell extracts to the same extent. Different NF1 family members showed different capacity to transactivate the LXRB gene promoter, but there was no difference between promoter alleles in NF1 induced transactivation activity.ConclusionVariations in the LXRB gene promoter may be part of the aetiology of T2D. However, the association between LXRB rs35463555 and rs17373080, and T2D are preliminary and needs to be investigated in additional larger cohorts. Common genetic variation in LXRA is unlikely to affect the risk of developing T2D or quantitative phenotypes related to glucose homeostasis.