scholarly journals Quinolone-DNA Interaction: Sequence-Dependent Binding to Single-Stranded DNA Reflects the Interaction within the Gyrase-DNA Complex

2003 ◽  
Vol 47 (3) ◽  
pp. 854-862 ◽  
Author(s):  
Christian G. Noble ◽  
Faye M. Barnard ◽  
Anthony Maxwell
Keyword(s):  
Metal Ions ◽  
Dna Cleavage ◽  
Specific Binding ◽  
Binding Mode ◽  
Dna Interaction ◽  
Dependent Manner ◽  
Sequence Dependence ◽  
Single Stranded Dna ◽  
Dna Complex

ABSTRACT We have investigated the interaction of quinolones with DNA by a number of methods to establish whether a particular binding mode correlates with quinolone potency. The specificities of the quinolone-mediated DNA cleavage reaction of DNA gyrase were compared for a number of quinolones. Two patterns that depended on the potency of the quinolone were identified. Binding to plasmid DNA was examined by measuring the unwinding of pBR322 by quinolones; no correlation with quinolone potency was observed. Quinolone binding to short DNA oligonucleotides was measured by surface plasmon resonance. The quinolones bound to both single- and double-stranded oligonucleotides in an Mg2+-dependent manner. Quinolones bound to single-stranded DNA with a higher affinity, and the binding exhibited sequence dependence; binding to double-stranded DNA was sequence independent. The variations in binding in the presence of metal ions showed that Mg2+ promoted tighter, more specific binding to single-stranded DNA than softer metal ions (Mn2+ and Cd2+). Single-stranded DNA binding by quinolones correlated with the in vitro quinolone potency, indicating that this mode of interaction may reflect the interaction of the quinolone with DNA in the context of the gyrase-DNA complex.

scholarly journals Molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120

2018 ◽  
Vol 475 (1) ◽  
pp. 151-168 ◽  
Author(s):  
Violeta C. Sein-Echaluce ◽  
María Carmen Pallarés ◽  
Anabel Lostao ◽  
Inmaculada Yruela ◽  
Adrián Velázquez-Campoy ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Specific Binding ◽  
Redox Homeostasis ◽  
Dna Interaction ◽  
Zinc Homeostasis ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Pcc 7120

FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium Anabaena PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner. In vitro, non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequences in FurB–DNA-binding affinity. Calorimetry studies showed that, in addition to one structural Zn(II), FurB is able to bind two additional Zn(II) per monomer and demonstrated the implication of cysteine C93 in regulatory Zn(II) coordination. The interaction of FurB with the second regulatory zinc occurred only under reducing conditions. While non-specific FurB–DNA interaction is Zn(II)-independent, the optimal binding of FurB to target promoters required loading of two regulatory zinc ions. Those results combined with site-directed mutagenesis and gel-shift assays evidenced that the redox state of cysteine C93 conditions the binding of the second regulatory Zn(II) and, in turn, modulates the affinity for a specific DNA target. Furthermore, differential spectroscopy studies showed that cysteine C93 could also be involved in heme coordination by FurB, either as a direct ligand or being located near the binding site. The results indicate that besides controlling zinc homeostasis, FurB could work as a redox-sensing protein probably modifying its zinc and DNA-binding abilities depending upon environmental conditions.

scholarly journals Mercury and Alzheimer’s Disease: Hg(II) Ions Display Specific Binding to the Amyloid-β Peptide and Hinder Its Fibrillization

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 44 ◽  
Author(s):  
Cecilia Wallin ◽  
Merlin Friedemann ◽  
Sabrina B. Sholts ◽  
Andra Noormägi ◽  
Teodor Svantesson ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Metal Ions ◽  
Amyloid Fibrils ◽  
Specific Binding ◽  
Amyloid Β ◽  
Binding Mode ◽  
Amyloid Β Peptide ◽  
Aβ Peptides

Brains and blood of Alzheimer’s disease (AD) patients have shown elevated mercury concentrations, but potential involvement of mercury exposure in AD pathogenesis has not been studied at the molecular level. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils. Aβ peptide fibrillization is known to be modulated by metal ions such as Cu(II) and Zn(II). Here, we study in vitro the interactions between Aβ peptides and Hg(II) ions by multiple biophysical techniques. Fluorescence spectroscopy and atomic force microscopy (AFM) show that Hg(II) ions have a concentration-dependent inhibiting effect on Aβ fibrillization: at a 1:1 Aβ·Hg(II) ratio only non-fibrillar Aβ aggregates are formed. NMR spectroscopy shows that Hg(II) ions interact with the N-terminal region of Aβ(1–40) with a micromolar affinity, likely via a binding mode similar to that for Cu(II) and Zn(II) ions, i.e., mainly via the histidine residues His6, His13, and His14. Thus, together with Cu(II), Fe(II), Mn(II), Pb(IV), and Zn(II) ions, Hg(II) belongs to a family of metal ions that display residue-specific binding interactions with Aβ peptides and modulate their aggregation processes.

scholarly journals Cu(II) and Ni(II) Complexes with New Tridentate NNS Thiosemicarbazones: Synthesis, Characterisation, DNA Interaction, and Antibacterial Activity

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Dorian Polo-Cerón
Keyword(s):  
Metal Complexes ◽  
Dna Cleavage ◽  
Thermal Analyses ◽  
Dna Interaction ◽  
Antimicrobial Activities ◽  
Tridentate Ligand ◽  
Bacterial Strains ◽  
Azomethine Group ◽  
Broth Microdilution Method

This paper reports the synthesis and detailed characterisation of copper(II) and nickel(II) complexes with tridentate thiosemicarbazone ligands H2L1 and H2L2 derived from 2-acetylpyrazine. The ligands and their metal complexes were characterised by different physicochemical techniques, including elemental and thermogravimetric analysis; UV-Vis, IR, 1H-NMR, and 13C-NMR spectroscopy; molar conductance measurements; and mass spectrometry. The crystal structure of the H2L1 ligand was determined by single crystal X-ray diffraction studies. The spectral data showed that the thiosemicarbazone behaves as an NNS tridentate ligand through the nitrogen atoms of the azomethine group and pyrazine ring and the sulphur atom of the thioamide group. Elemental and thermal analyses indicated that the obtained metal complexes had a 1 : 1 stoichiometry (metal-ligand). The interactions between these complexes and calf thymus DNA (CT-DNA) were studied by electronic absorption and viscosity measurements. The activities of these compounds against oxidative DNA cleavage were examined by agarose gel electrophoresis. Cu(II) and Ni(II) complexes can wind DNA strands through groove interactions and promote strand breakage of the plasmid pmCherry under oxidative stress conditions. Moreover, all the complexes could interact more strongly with DNA than could with the free ligands. Finally, the antibacterial activities of the ligands and their complexes were determined by in vitro tests against Gram-positive bacterial strains (S. aureus ATCC 25923, L. monocytogenes ATCC 19115, and B. cereus ATCC 10876) and Gram-negative bacterial strains (E. coli ATCC 25922, S. typhimurium ATCC 14028, and K. pneumoniae ATCC BAA-2146) using the broth microdilution method. The metal complexes showed greater antimicrobial activities than the precursor ligands against some of the microorganisms.

scholarly journals Antibody blockade of Dectin-2 suppresses house dust mite-induced Th2 cytokine production in dendritic cell- and monocyte-depleted peripheral blood mononuclear cell co-cultures from asthma patients

2019 ◽  
Vol 26 (1) ◽  
Author(s):  
Ming-Han Chen ◽  
Ming-Ting Huang ◽  
Wen-Kuang Yu ◽  
Shinn-Shing Lee ◽  
Jia-Horng Wang ◽  
...  
Keyword(s):  
House Dust ◽  
House Dust Mite ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Specific Binding ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Dust Mite ◽  
Der P 2

Abstract Background Dectin-2, which is a C-type lectin, interacts with the house dust mite (HDM) Dermatophagoides pteronyssinus allergen. This study aimed to investigate whether Dectin-2 blockade by antagonistic monoclonal antibodies (MoAbs) attenuates HDM-induced allergic responses. Methods Two anti-Dectin-2 MoAbs were generated and validated for specific binding to Dectin-2 Fc fusion protein (Dectin-2.Fc) and inhibition of Dectin-2.Fc/HDM interaction. Patients with asthma exhibiting high titers of anti-D. pteronyssinus IgE were enrolled. Peripheral blood mononuclear cells with depleted CD14+ monocytes were obtained from these patients and co-cultured with autologous monocyte-derived conventional dendritic cells in the presence of D. pteronyssinus or its group 2 allergens (Der p 2). Interleukin (IL)-5 and IL-13 levels in the culture supernatants were determined using ELISA in the presence or absence of anti-Dectin-2 MoAbs. Results Two MoAbs, 6A4G7 and 17A1D10, showed specific binding to recombinant Dectin-2.Fc and inhibited HDM binding to Dectin-2.Fc. Both anti-Dectin-2 MoAbs inhibited IL-5 and IL-13 production in co-cultures with Der p 2 stimulation in a dose-dependent manner. 6A4G7 and 17A1D10 (3 μg/mL) significantly inhibited Der p 2-induced (3 μg/mL) IL-5 production by 69.7 and 86.4% and IL-13 production by 84.0 and 81.4%, respectively. Moreover, this inhibitory effect of the two MoAbs remained significant in the presence of D. pteronyssinus. Conclusions Anti-Dectin-2 MoAbs significantly inhibited HDM-induced allergic responses in vitro and therefore have the potential to become therapeutic agents in mite-induced allergic diseases.

Effects of corticostatin-I on rat adrenal cells in vitro

1990 ◽  
Vol 125 (2) ◽  
pp. 287-292 ◽  
Author(s):  
T. Tominaga ◽  
J. Fukata ◽  
Y. Naito ◽  
Y. Nakai ◽  
S. Funakoshi ◽  
...  
Keyword(s):  
Specific Binding ◽  
Corticosterone Level ◽  
Inhibitory Effect ◽  
Zona Glomerulosa ◽  
Dependent Manner ◽  
Minimum Effective Concentration ◽  
Adrenal Cells ◽  
Aldosterone Production ◽  
The Media

ABSTRACT We have examined the mechanism by which corticostatin-I (CS-I) acts to attenuate ACTH-induced steroidogenesis in rat adrenal cells. CS-I inhibited ACTH-induced corticosterone production in a dosedependent manner, without any effects on the basal corticosterone level in adrenal cells. When the cells were stimulated by 100 pg ACTH/ml, the minimum effective concentration of CS-I was 100 ng/ml, and 0.3–1.0 μg CS-I/ml produced a 50% reduction of the stimulated corticosterone production. The inhibitory effect of CS-I on ACTH-stimulated corticosterone production became apparent within 15 min of incubation, and the effect was reversed quickly by the removal of CS-I from the media. CS-I had no effect on angiotensin II-stimulated aldosterone production by adrenal zona glomerulosa cells. CS-I also did not affect cyclic AMP- or forskolin-stimulated corticosterone production. In an in-vitro binding study using 125I-labelled CS-I, CS-I showed considerable specific binding to rat adrenal cells, and the binding competed with ACTH in a dose-dependent manner. These experiments suggest that CS-I competes with ACTH on their binding sites and exerts an inhibitory effect on the adrenal cells. Journal of Endocrinology (1990) 125, 287–292

Apoptosis Inducing Effects of Thymus linearis Methanolic Extract in HCT-116 Cells and LC-MS Chemical Profiling of Its Active Constituents

2021 ◽  
Vol 11 ◽  
Author(s):  
Rohina Bashir ◽  
Ovais Zargar ◽  
Qazi Parvaiz ◽  
Rabia Hamid
Keyword(s):  
Cell Lines ◽  
Antiproliferative Activity ◽  
Dna Cleavage ◽  
Methanolic Extract ◽  
Dependent Manner ◽  
Regulation Of Expression ◽  
Anti Cancer ◽  
Hct 116 ◽  
Hct 116 Cells

Background: Cancer is one of the major problems at present, to which vast research is being dedicated to find effective remedy. Medicinal plants are endowed with numerous molecules that could be effective in multiple diseases including cancer. Thymus linearis, being rich in phenols, terpenoid, and flavonoids have potential to provide anti-cancer entities. Methods: The extracts of Thymus linearis were investigated for in vitro anticancer activity using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay on a panel of cancer cell lines. The cellular and nuclear morphology was studied using microscopic techniques. Agarose gel electrophoresis was used for DNA fragmentation analysis. Protein expression was determined by western-blotting. LC-MS was used for phytochemical identification. Results: Among all the extracts, Thymus linearis methanolic (TLM) extract was found to exhibit antiproliferative activity on cell lines to varied degrees. TLM was found to be most potent against HCT-116 with an IC50 of 158μg/ml after 48hrs treatment, while being nontoxic to HEK-293 and FR-2 cells under similar concentrations. TLM decreased clonogenic potential of HCT-116 cells. It induced cell shrinkage, membrane blebbing and nuclear fragmentation characteristic of apoptotic in a dose dependent manner in HCT-116 cells. Prominent internucleosomal DNA cleavage was observed in HCT-116 cells after 48hrs TLM treatment. Western blot analysis revealed the up regulation of expression of Bax, caspases 9 and caspases 3 and downregulation of Bcl-2 proteins. The LC-MS data revealed the presence of Salvianolic acid H, Synparvolide C, Thymuside A and Jasmonic acid; 12-Hydroxy, O-β-D-glucopyranoside and polyphenolic flavonoids to which antiproliferative activity can be attributed. Conclusion: The results suggest that Thymus linearis methanolic extract could be valuable source of anti-cancer agents.

scholarly journals In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter.

1991 ◽  
Vol 11 (4) ◽  
pp. 2162-2168 ◽  
Author(s):  
E Dubois ◽  
F Messenguy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Binding ◽  
Regulatory Element ◽  
Regulatory Proteins ◽  
Arginine Metabolism ◽  
Mobility Shift ◽  
Catabolic Genes ◽  
Gel Mobility Shift ◽  
Dna Complex

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

scholarly journals Novel DNA Bis-intercalation by MLN944, a Potent Clinical Bisphenazine Anticancer Drug

2004 ◽  
Vol 279 (44) ◽  
pp. 46096-46103 ◽  
Author(s):  
Jixun Dai ◽  
Chandanamalie Punchihewa ◽  
Prakash Mistry ◽  
Aik Teong Ooi ◽  
Danzhou Yang
Keyword(s):  
Dna Binding ◽  
Anticancer Drug ◽  
Topoisomerase I ◽  
Specific Binding ◽  
Binding Mode ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Phase I Clinical Trials ◽  
Mobility Shift ◽  
Dna Sequence Specificity

The new bisphenazine anticancer drug MLN944 is a novel cytotoxic agent with exceptional anti-tumor activity against a range of human and murine tumor models both invitroand invivo. MLN944 has recently entered Phase I clinical trials. Despite the structural similarity with its parent monophenazine carboxamide and acridine carboxamide anticancer compounds, MLN944 appears to work by a distinct mechanism of inhibiting DNA transcription rather than the expected mechanism of topoisomerase I and II inhibition. Here we present the first NMR structure of MLN944 complexed with d(ATGCAT)2DNA duplex, demonstrating a novel binding mode in which the two phenazine rings bis-intercalate at the 5′-TpG site, with the carboxamide amino linker lying in the major groove of DNA. The MLN944 molecule adopts a significantly unexpected conformation and side chain orientation in the DNA complex, with the N10 on the phenazine ring protonated at pH 7. The phenazine chromophore of MLN944 is very well stacked with the flanking DNA base pairs using the parallel base-stacking intercalation binding mode. The DNA sequence specificity and the groove recognition of MLN944 binding is determined by several site-specific hydrogen bond interactions with the central G:C base pair as well as the favorable stacking interactions with the 5′-flanking thymine. The specific binding site of MLN944 is known to be recognized by a number of important transcription factors. Our electrophoretic gel mobility shift assay results demonstrated that the c-Jun DNA binding to the AP-1 site is significantly inhibited by MLN944 in a dose-dependent manner. Thus, the exceptional biological activity of MLN944 may be due to its novel DNA binding mode leading to a unique mechanism of action.

scholarly journals The ClgR Protein Regulates Transcription of the clpP Operon in Bifidobacterium breve UCC 2003

2005 ◽  
Vol 187 (24) ◽  
pp. 8411-8426 ◽  
Author(s):  
Marco Ventura ◽  
Ziding Zhang ◽  
Michelle Cronin ◽  
Carlos Canchaya ◽  
John G. Kenny ◽  
...  
Keyword(s):  
Promoter Region ◽  
Specific Binding ◽  
Three Dimensional ◽  
Binding Mode ◽  
Dna Interaction ◽  
Dimensional Structure ◽  
Mobility Shift ◽  
Significant Similarity ◽  
Bifidobacterium Breve

ABSTRACT Five clp genes (clpC, clpB, clpP1, clpP2, and clpX), representing chaperone- and protease-encoding genes, were previously identified in Bifidobacterium breve UCC 2003. In the present study, we characterize the B. breve UCC 2003 clpP locus, which consists of two paralogous genes, designated clpP1 and clpP2, whose deduced protein products display significant similarity to characterized ClpP peptidases. Transcriptional analyses showed that the clpP1 and clpP2 genes are transcribed in response to moderate heat shock as a bicistronic unit with a single promoter. The role of a clgR homologue, known to control the regulation of clpP gene expression in Streptomyces lividans and Corynebacterium glutamicum, was investigated by gel mobility shift assays and DNase I footprint experiments. We show that ClgR, which in its purified form appears to exist as a dimer, requires a proteinaceous cofactor to assist in specific binding to a 30-bp region of the clpP promoter region. In pull-down experiments, a 56-kDa protein copurified with ClgR, providing evidence that the two proteins also interact in vivo and that the copurified protein represents the cofactor required for ClgR activity. The prediction of the ClgR three-dimensional structure provides further insights into the binding mode of this protein to the clpP1 promoter region and highlights the key amino acid residues believed to be involved in the protein-DNA interaction.

scholarly journals In Vivo Requirement of Activator-Specific Binding Targets of Mediator

2000 ◽  
Vol 20 (23) ◽  
pp. 8709-8719 ◽  
Author(s):  
Jin Mo Park ◽  
Hye-Suk Kim ◽  
Sang Jun Han ◽  
Moon-Sun Hwang ◽  
Young Chul Lee ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Specific Binding ◽  
Gene Activation ◽  
Dna Interaction ◽  
Activation Process ◽  
Binding Region ◽  
Physiological Conditions ◽  
Functional Analyses

ABSTRACT There has been no unequivocal demonstration that the activator binding targets identified in vitro play a key role in transcriptional activation in vivo. To examine whether activator-Mediator interactions are required for gene transcription under physiological conditions, we performed functional analyses with Mediator components that interact specifically with natural yeast activators. Different activators interact with Mediator via distinct binding targets. Deletion of a distinct activator binding region of Mediator completely compromised gene activation in vivo by some, but not all, transcriptional activators. These demonstrate that the activator-specific targets in Mediator are essential for transcriptional activation in living cells, but their requirement was affected by the nature of the activator-DNA interaction and the existence of a postrecruitment activation process.

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