1, 4-naphthoquinone, a plant-based quinone derivative, has gained much attention for its effectiveness against several biofilm-linked diseases. The biofilm inhibitory effect of 1, 4-naphthoquinone against Staphylococcus aureus has already been reported in our previous study. We observed that the extracellular DNA (eDNA) could play an important role in holding the structural integrity of the biofilm. Hence, in this study, efforts have been directed to examine the possible interactions between 1, 4-naphthoquinone and DNA. An in silico analysis indicated that 1, 4-naphthoquinone could interact with DNA through intercalation. To validate the same, UV-Vis spectrophotometric analysis was performed in which a hypochromic shift was observed when the said molecule was titrated with calf-thymus DNA (CT-DNA). Thermal denaturation studies revealed a change of 8°C in the melting temperature (Tm) of CT-DNA when complexed with 1, 4-naphthoquinone. The isothermal calorimetric titration (ITC) assay revealed a spontaneous intercalation between CT-DNA and 1, 4-naphthoquinone with a binding constant of 0.95 ± 0.12 × 108. Furthermore, DNA was run through an agarose gel electrophoresis with a fixed concentration of ethidium bromide and increasing concentrations of 1, 4-naphthoquinone. The result showed that the intensity of ethidium bromide-stained DNA got reduced concomitantly with the gradual increase of 1, 4-naphthoquinone suggesting its intercalating nature. To gain further confidence, the pre-existing biofilm was challenged with ethidium bromide wherein we observed that it could also show biofilm disintegration. Therefore, the results suggested that 1, 4-naphthoquinone could exhibit disintegration of the pre-existing biofilm of Staphylococcus aureus through eDNA intercalation.
- MeSH
- Biofilms MeSH
- DNA pharmacology MeSH
- Ethidium pharmacology MeSH
- Humans MeSH
- Naphthoquinones * pharmacology MeSH
- Staphylococcal Infections * MeSH
- Staphylococcus aureus genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.
- MeSH
- Hep G2 Cells MeSH
- DNA-Binding Proteins metabolism MeSH
- Doxorubicin MeSH
- Ethidium MeSH
- GTP Phosphohydrolases metabolism MeSH
- Mitochondria, Liver metabolism MeSH
- Humans MeSH
- DNA, Mitochondrial metabolism MeSH
- Mitochondrial Proteins metabolism MeSH
- Tumor Suppressor Protein p53 metabolism MeSH
- DNA Damage MeSH
- Microtubule-Associated Proteins metabolism MeSH
- Transcription Factors metabolism MeSH
- Mitochondrial Membrane Transport Proteins metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
In this work, a new partial filling affinity capillary electrophoresis (PF-ACE) method has been developed and applied to investigation of non-covalent molecular interactions between double stranded DNA oligonucleotide (Dickerson dodecamer) and classical DNA intercalator ligand-ethidiumbromide (EtBr) or oligophenylene derivatives-based potential new type of DNA ligands. Binding constants of DNA-ligand complexes were determined from the dependence of migration time changes of DNA oligomer (applied as analyte) on the length of ligand zones introduced beforehand as plugs of various lengths (0-75mm with 12.5mm step) in hydroxypropylcellulose coated fused silica capillary of 50/375μm I.D./O.D. and 400/300mm total/effective length. PF-ACE experiments were performed in two background electrolytes, Tris-borate, pH 8.0, ionic strength 14.3mM (BGE1), and sodium phosphate, pH 7.5, ionic strength 133mM (BGE2). Binding constants of DNA-EtBr complex (ca 15300L/mol in the BGE1 and 4200L/mol in the BGE2) were found to be significantly higher than those of DNA complexes with oligophenylene derivatives (ca 2200-3600L/mol in the BGE1 and 1600-2300L/mol in the BGE2).
Leptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n=11), transport (n=6), membrane structure (n=5), stress response (n=2), DNA damage repair (n=1), and other processes (n=3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenic L. interrogans.
- MeSH
- Anti-Infective Agents pharmacology MeSH
- Drug Resistance, Bacterial genetics MeSH
- Bacterial Proteins genetics MeSH
- Biological Transport MeSH
- Ethidium pharmacology MeSH
- Phenotype MeSH
- Gene Library MeSH
- Mutagenesis, Insertional MeSH
- Leptospira genetics physiology MeSH
- Membrane Transport Proteins genetics MeSH
- Microbial Sensitivity Tests MeSH
- Operon genetics MeSH
- Drug Tolerance genetics MeSH
- DNA Transposable Elements genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Apoptóza je typ programované buněčné smrti (typ I), který je nezbytný pro správný vývoj organizmu a tkáňovou homeostázu. Její průběh může být určen dvěma signálními drahami – vnější (receptorovou) dráhou řízenou receptory smrti a vnitřní (mitochondriální) apoptotickou dráhou, kde klíčovou roli plní mitochondrie. Mitochondrie jsou důležité buněčné organely s nepostradatelnými funkcemi pro život buňky, jako je např. tvorba energie ve formě molekul ATP (adenosintrifosfátu). Mitochondriální buněčná smrt je charakteristická změnou transmembránového potenciálu a permeabilizací vnější mitochondriální membrány. Mitochondrie jsou elektronegativní organely a depolarizace mitochondriální membrány je důležitá pro uvolnění proapoptotických signálů. Narušená regulace mitochondriální buněčné smrti se může podílet na patogenezi různých onemocnění, včetně rakoviny. Mitochondrie jsou také zdrojem reaktivních kyslíkových radikálů, iontů Ca2+ a proteinů ovlivňujících procesy iniciace a progrese nádorů nezávisle na indukci apoptózy. Současné studie se zaměřují na výzkum mitochondriálního membránového potenciálu a kyslíkových radikálů, které modulují různé signální dráhy uvnitř buňky a vymezení jejich významu v kancerogenezi, případně v léčbě onkologických pacientů. Monitorování apoptotických markerů, jako je stav mitochondriálního membránového potenciálu a určení hladiny reaktivních kyslíkových radikálů ve vzorcích nádorových pacientů, má prediktivní hodnotu pro výstup léčebných protokolů.
Apoptosis is type I programmed cell death, a process that is essential for development and tissue homeostasis. It is a prevalent form of cell death and it proceeds via two signaling pathways – external (receptor pathway) triggered by death receptors and intrinsic (mitochondrial) apoptotic pathway with major involvement of mitochondria. Mitochondria are important cellular organelles producing energy stored in molecules of adenosine triphosphate that are essential for cell survival. The mitochondrial cell death is characterized by permeabilization of the mitochondrial outer membrane and dissipation of the transmembrane potential. Mitochondria are electronegative organelles and depolarization of the mitochondrial membrane is important for the release of proapoptotic signals. Aberrant control of the mitochondrial cell death might contribute to several diseases including cancer. Mitochondria are also a source of reactive oxygen species, Ca2+ ions and other proteins that affect processes important for the initiation and progression of tumors independently of apoptosis. Current studies focus on research of mitochondrial membrane potential and reactive oxygen species modulating various signaling pathways within the cell, their importance in carcinogenesis, and in treatment of oncological patients. Monitoring of the apoptotic markers, such as the mitochondrial membrane potential (MMP), and the level of reactive oxygen species in samples of oncological patients has a predictive value for the output of treatment protocols. Key words: mitochondria – flow cytometry – apoptosis – free radicals – mitochondrial membrane potential This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and IntegRECAMO CZ.1.07/2.3.00/20.0097). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 13. 1. 2014 Accepted: 11. 4. 2014
- MeSH
- Apoptosis * physiology MeSH
- Cell Death physiology MeSH
- Ethidium analogs & derivatives diagnostic use MeSH
- Fluoresceins diagnostic use MeSH
- Fluorescent Dyes diagnostic use MeSH
- Humans MeSH
- Membrane Potential, Mitochondrial * MeSH
- Mitochondrial Membranes MeSH
- Mitochondria MeSH
- Flow Cytometry * methods MeSH
- Reactive Oxygen Species * analysis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn(2+). All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.
- MeSH
- Epithelial Cells metabolism MeSH
- Ethidium analogs & derivatives MeSH
- Fluoresceins MeSH
- Rabbits MeSH
- Rats MeSH
- Methods MeSH
- Molecular Probe Techniques MeSH
- Myocardium metabolism MeSH
- Lens, Crystalline cytology MeSH
- Reactive Oxygen Species analysis MeSH
- Rhodamines MeSH
- Free Radicals analysis MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Rats MeSH
- Animals MeSH
- Publication type
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
[Fe(2)L(3)](4+) (L = C(25)H(20)N(4)) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe(2)L(3)](4+), we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine-pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe(2)(L-CF(3))(3)](4+) and [Fe(2)(L-Ph)(3)](4+) results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.
- MeSH
- Deoxyribonuclease I MeSH
- DNA Footprinting MeSH
- DNA chemistry metabolism MeSH
- Ethidium chemistry MeSH
- Financing, Organized MeSH
- Binding, Competitive MeSH
- Nucleic Acid Conformation MeSH
- Pyridines chemistry MeSH
- DNA Restriction Enzymes metabolism MeSH
- Base Sequence MeSH
- Stereoisomerism MeSH
- DNA, Superhelical chemistry MeSH
- Ferrous Compounds chemistry MeSH
This work is the first in-depth study of osmium binding to DNA and confirms the pharmacological activity of a new class of anticancer metallodrugs. We investigated the interactions between the potential biological target DNA and four osmium(II) arene complexes, of the type [(eta 6-arene)Os(LL)Cl]n+, where arene = biphenyl or p-cymene and LL = ethylenediamine, picolinate, or oxinate in an effort to understand their mechanism of action. Most notably we show that these complexes bind to DNA. DNA adducts of the OsII complexes that exhibit promising cytotoxic effects in ovarian tumor cell lines largely distort its conformation. The data are consistent with DNA binding of the complexes containing biphenyl as the arene ligand that involves combined coordination to guanine residues and noncovalent interactions between the arene ligand and DNA. The results also indicate both a mechanism of action and a detoxification mechanism for OsII arene compounds different from those of cisplatin.
- MeSH
- DNA Adducts chemistry MeSH
- Cell-Free System MeSH
- Drug Resistance, Neoplasm MeSH
- Cisplatin chemistry MeSH
- DNA chemistry metabolism MeSH
- Ethidium MeSH
- Financing, Organized MeSH
- Fluorescent Dyes MeSH
- Spectrometry, Fluorescence MeSH
- Intercalating Agents MeSH
- Humans MeSH
- Molecular Conformation MeSH
- Cell Line, Tumor MeSH
- Oligonucleotides chemistry MeSH
- Organometallic Compounds chemistry metabolism MeSH
- Osmium MeSH
- Antineoplastic Agents chemistry metabolism MeSH
- Drug Screening Assays, Antitumor MeSH
- DNA, Superhelical chemistry MeSH
- Transition Temperature MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
Metallosupramolecular chemistry was used to design a new class of synthetic agents, namely, tetracationic supramolecular cylinders, that bind strongly and noncovalently in the major groove of DNA. To gain additional information on interactions of the cylinders with DNA we explored DNA unwinding and sequence-specific binding properties, as well as DNA photonuclease activity of ruthenium(II) metallosupramolecular cylinder [Ru(2)L(3)](4+), where L is a bis-pyridylimine ligand. We found that [Ru(2)L(3)](4+) unwinds negatively supercoiled plasmid DNA and exhibits binding preference to regular alternating purine-pyrimidine sequences in a similar way to the [Fe(2)L(3)](4+) analogue. Photocleavage studies showed that, unlike [Fe(2)L(3)](4+), [Ru(2)L(3)](4+) induces single-strand breaks on irradiation by visible and UVA light and cleaves DNA mainly at guanine residues contained preferentially in regularly alternating purine-pyrimidine nucleotides. As [Ru(2)L(3)](4+) binds and cleaves DNA in a sequence-dependent manner, it may provide a useful tool for basic and applied biology, such as for controlled manipulation of the genome.
Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.
- MeSH
- Antimycin A analogs & derivatives pharmacology MeSH
- Cell Respiration MeSH
- Electron Spin Resonance Spectroscopy MeSH
- Ethidium analogs & derivatives chemistry MeSH
- Ferricyanides pharmacology MeSH
- Financing, Organized MeSH
- Glycerolphosphate Dehydrogenase metabolism MeSH
- Glycerophosphates metabolism MeSH
- Adipose Tissue, Brown enzymology drug effects ultrastructure MeSH
- Cricetinae MeSH
- Mitochondria enzymology metabolism drug effects MeSH
- Reactive Oxygen Species analysis metabolism MeSH
- Electron Transport Complex III metabolism MeSH
- Oxygen Consumption MeSH
- Electron Transport MeSH
- Ubiquinone metabolism MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Male MeSH
- Animals MeSH