Presence Of Ethidium Bromide Research Articles

DNA interaction, in vitro antimicrobial and SOD-like activity of copper(II) complexes with norfloxacin and terpyridines

The novel octahedral copper(II) complexes with the second generation fluoroquinolone, norfloxacin and terpyridine derivatives were prepared and characterized. The antimicrobial efficiency of the complexes were tested on five different microorganisms and showed good biological activity. Viscosity measurement and absorption titration were employed to determine the mode of binding of complexes with DNA whereas the cleavage efficacy of the complexes towards pUC19 DNA was determined by electrophoresis in presence of ethidium bromide. SOD mimic behaviour was actively sought for clinical and mechanistic purposes under a nonenzymatic system (NBT/NADH/PMS), and was found to have good antioxidant activity.

Human Mre11/Human Rad50/Nbs1 and DNA Ligase IIIα/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.

Ternary copper(II) complexes of levofloxacin and phenanthroline derivatives: In-vitro antibacterial, DNA interactions, and SOD-like activity

A series of ternary copper(II) complexes have been derived using levofloxacin and five phenanthroline derivatives. Complexes were characterized using infrared spectroscopy, Thermogravimetric (TG)-analysis, fast atom bombardment mass spectroscopy and reflectance spectra. Synthesized complexes exhibit the only d-d band at ∼ 666 nm points toward a distorted square pyramidal geometry at metal centre with one unpaired electron responsible for paramagnetic behaviour of whole moiety. Binding behaviour of the complexes toward Herring Sperm DNA were determined using ultraviolet-Vis (UV-Vis) absorption titration and viscometric titration experiment, where as the cleavage efficacy of the complexes toward pUC19 DNA was determined by electrophoresis in presence of ethidium bromide. Complexes exhibit superoxide dismutase–like activity with their IC50 values ranging from 0.7917 to 1.7432 µM.

Mitochondrial DNA Depletion Induces Radioresistance by Suppressing G2Checkpoint Activation in Human Pancreatic Cancer Cells

We hypothesized that mitochondrial function regulates cell cycle checkpoint activation and radiosensitivity. Human pancreatic tumor cells (MiaPaCa-2, rho(+)) were depleted of mitochondrial DNA (rho degrees ) by culturing cells in the presence of ethidium bromide. Depletion of mitochondrial DNA was verified by PCR amplification of total DNA using primer pairs specific for mitochondrial DNA. Loss of mitochondrial DNA decreased plating efficiency and the percentage of cells in S phase. Exponential cultures were irradiated with 2, 4 and 6 Gy (dose rate: 0.83 Gy/min) of ionizing radiation and harvested for determination of cell viability, growth and cell cycle phase distributions. Rho degrees cells were radioresistant compared to rho(+) cells, with a dose-modifying factor (DMF) of 1.6. Although cell growth was significantly inhibited in irradiated rho(+) cells compared to unirradiated control cells, the inhibition in Rho degrees cells was minimal. In addition, mitochondrial DNA depletion suppressed radiation-induced G(2) checkpoint activation, which was accompanied by increases in both cyclin B1 and CDK1. These results suggest that mitochondrial function may regulate cell cycle checkpoint activation and radiosensitivity in pancreatic cancer cells.

Cellular Responses to Cancer Chemopreventive Agent D,L-Sulforaphane in Human Prostate Cancer Cells Are Initiated by Mitochondrial Reactive Oxygen Species

Present study was undertaken to elucidate the mechanism of cellular responses to D,L-sulforaphane (SFN), a highly promising cancer chemopreventive agent. Mitochondrial DNA deficient Rho-0 variants of LNCaP and PC-3 cells were generated by culture in the presence of ethidium bromide. Apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation and activation of caspase-3. Immunoblotting was performed to determine the expression of apoptosis- and cell cycle-regulating proteins. Generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and cell cycle distribution were measured by flow cytometry. The Rho-0 variants of LNCaP and PC-3 cells were significantly more resistant to SFN-induced ROS generation, apoptotic DNA fragmentation, disruption of MMP, cytosolic release of cytochrome c, and G2/M phase cell cycle arrest compared with corresponding wild-type cells. SFN-induced autophagy, which serves to protect against apoptotic cell death in PC-3 and LNCaP cells, was also partially but markedly suppressed in Rho-0 variants compared with wild-type cells. SFN statistically significantly inhibited activities of mitochondrial respiratory chain enzymes in LNCaP and PC-3 cells. These results indicate, for the first time, that mitochondria-derived ROS serve to initiate diverse cellular responses to SFN exposure in human prostate cancer cells.

Small-Molecule Binding to DNA under Tension and Twist Studied by Magnetic Tweezers

DNA-binding small molecules are present in the cellular environment and are ubiquitously used in biochemistry and biotechnology. Here, we use single molecule magnetic tweezers experiments to study the effect of small-molecule ligands on DNA mechanical properties.Using the magnetic tweezers ability to control both the applied stretching force and torque, we have systematically characterized the mechanical properties of DNA in the presence of Ethidium Bromide (EtBr), a well-known intercalator, and Netropsin, an anti-microbial drug and known minor groove binder. In addition, we have characterized the interactions of Topotecan, a clinically used anti-tumor drug, with bare DNA.Our results show a lengthening of DNA upon EtBr intercalation to an extension of ∼1.5 times the initial contour length and a decrease in DNA twist by 29 +- 3 degrees per intercalation event, in agreement with previous estimates from bulk experiments. Further effects of intercalation are a stabilization of the double strand under high forces and negative torques and a decrease in both the bending and twisting persistence lengths. In contrast, minor groove binding by Netropsin does not change the contour or persistence length significantly, but increases the twist per base of the DNA.For Topotecan, both intercalative and minor-groove binding have been postulated. Our magnetic tweezers results indicate that drug binding at > 50 micromolar concentrations has consequences qualitatively similar to the changes observed for EtBr, in support of a intercalative binding mode for Topotecan.These results point to important consequences of intercalative ligand binding for DNA torsional behavior, which we characterized at the single-molecule level using magnetic tweezers. Ultimately, such insights can help elucidate the effects of small molecule drugs in the cellular environment and their interference with DNA transcription and replication.

Modulation of Photo-oxidative DNA Damage by Cationic Surfactant Complexation

The natural packaging of DNA in the cell by histones provides a particular environment affecting its sensitivity to oxidative damage. In this work, we used the complexation of DNA by cationic surfactants to modulate the conformation, the dynamics, and the environment of the double helix. Photo-oxidative damage initiated by benzophenone as the photosensitizer on a plasmid DNA complexed by dodecyltrimethylammonium chloride (DTAC), tetradecyltrimethylammonium chloride (TTAC), cetyltrimethyammonium chloride (CTAC) and bromide (CTAB) was detected by agarose gel electrophoresis. By fluorescent titration in the presence of ethidium bromide (EB) and agarose gel electrophoresis, we experimentally confirmed the complexation diagrams with a critical aggregation concentration on DNA matrix (CAC DNA) delimiting two regions of complexation, according to the DNA-phosphate concentration. The study of the photo-oxidative damage shows, for the first time, a direct correlation between the DNA complexation by these surfactants and the efficiency of DNA cleavage, with a maximum corresponding to the CAC DNA for DTAC and CTAC, and to DNA neutralization for CTAC and CTAB. The localization of a photosensitizer having low water solubility, such as benzophenone, inside the hydrophobic domains formed by the surfactant aggregated on DNA, locally increases the photoinduced cleavage by the free radical oxygen species generated. The inefficiency of a water-soluble quencher of hydroxyl radicals, such as mannitol, confirmed this phenomenon. The detection of photo-oxidative damage constitutes a new tool for investigating DNA complexation by cationic surfactants. Moreover, highlighting the drastically increased sensitivity of a complexed DNA to photo-oxidative damage is of crucial importance for the biological use of surfactants as nonviral gene delivery systems.

Yeast Pgc1p (YPL206c) Controls the Amount of Phosphatidylglycerol via a Phospholipase C-type Degradation Mechanism

The product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces cerevisiae displays homology to bacterial and mammalian glycerophosphodiester phosphodiesterases. Deletion of PGC1 causes an accumulation of the anionic phospholipid, phosphatidylglycerol (PG), especially under conditions of inositol limitation. This PG accumulation was not caused by increased production of phosphatidyl-glycerol phosphate or by decreased consumption of PG in the formation of cardiolipin, the end product of the pathway. PG accumulation in the pgc1Delta strain was caused rather by inactivation of the PG degradation pathway. Our data demonstrate an existence of a novel regulatory mechanism in the cardiolipin biosynthetic pathway in which Pgc1p is required for the removal of excess PG via a phospholipase C-type degradation mechanism.

Detection of schistosomes polymerase chain reaction amplified DNA by oligochromatographic dipstick

The applications of highly specific and sensitive molecular techniques based on polymerase chain reaction (PCR) have constituted a valuable tool for the diagnosis of schistosomiasis and also for the detection of schistosome infections in the snail intermediate hosts. The common method of detecting PCR amplicons is gel electrophoresis in the presence of ethidium bromide, a carcinogen, which is followed by UV transillumination. Other methods, which are available for detecting PCR products, are real-time PCR, PCR–enzyme-linked immunosorbent assay (PCR–ELIZA) and mass spectrometry but they are cumbersome while they are sometimes complex and expensive. Therefore, a simple method of PCR product detection would be a welcome idea and a most valuable tool particularly in disease endemic countries with limited research facilities and resources. In this study, we applied a simple and rapid method for the detection of Schistosoma haematobium and Schistosoma mansoni PCR amplified DNA products using oligochromatographic (OC) dipstick. The amplicons are visualized by hybridization with a gold conjugated probe, while a control for the chromatographic migration is incorporated in the assay. The lower detection limit observed was 10fg of genomic DNA from each of the two species, while the dipstick was also specific for each of the species used in this study.

Creating line defects with a single micro-sphere width in a DNA-linked 2D colloidal crystal array using a pulsed laser

A creation of micron-size narrow line defects in 2D colloidal crystal array was successfully accomplished using a focused pulsed laser. The 2D colloidal crystal arrays were obtained by a convective assembly of polystyrene latex micro-spheres (1.8 μm) and then immobilized onto the glass substrate by DNA hybridization process. To release a micro-sphere from the crystal, a focused laser beam was utilized to de-hybridize the DNA by raising the temperature above its melting point. From previous studies (Geiss E, Fabrication and defect design in two-dimensional colloidal photonic crystals. Ph.D dissertation thesis, 2003; Kim et al., Mater Sci Eng 26:1401–1407, 2006; Geiss et al., sudmitted), using a continuous argon ion laser, 514 nm, line-writing in colloidal crystal with 2–4 micro-sphere widths were achieved. In this study using the Nd:YAG pulsed laser’s second harmonic, 532 nm, line-writing of 1 micro-sphere width was accomplished. The presence of EB (ethidium bromide) dye that intercalates into the ds-DNA strands that bind the micro-spheres to the substrate facilitated the micro-sphere releasing process by reducing the laser energy required to release a micro-sphere. Factors that influenced controlled defect insertion were the medium containing the colloidal crystal, the dyes imposed on the crystal and the self-assembled micro-sphere groups. The most effective medium condition were determined at 1 mM global EB dye in 0.1 M NaCl for removing an individual micro-sphere in single micro-spheres, groups of micro-spheres and the close-packed arrays. Single micro-sphere width simple curves and corners in the photonic crystals were then created.

Microfluorimetric analysis of dynamics of genomic changes in Chinese hamster fibroblasts CHL V-79 RJK with multiple drug resistance

Results of karyological analysis of cells CHL V-79 RJK selected for resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (line Vebr-5) were compared with the data of microfluorimetric determination of DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (ADM) in the form of homogenously or differentially stained regions (HSRs and DSRs, respectively) in two chromosomes (Z1 and Z6, loci 1 p29-31 and 1q26, respectively). HSRs in Z6, in the region of localization of the wild type of gene mdr, had unstable length and structure characteristic of morphological markers of amplification of genes of the family mdr. During long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSRs in Z6 increased. Results of microfluorimetric analysis of Vebr-5 at the 11th passage have shown an increase in the DNA content not only in chromosomes Z1 and Z6 marked by HSRs, but also in three chromosomes (Z5, Z12 and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed non-random changes in the DNA content in four more chromosomes: an increase in Z14, while a decrease in chromosomes 8, Z7, and Z9. A decrease of the DNA content in chromosomes is considered to be a result of a partial loss of genetic material, while its increase is a result of its translocation and (or) amplification. Coefficient of variation of the DNA content changes for large chromosomes amounted to about 9%. while for small chromosomes it is about 26%, which indicates that small chromosomes have greater potential for instability than the large ones. The data obtained not only confirm, but also enlarge the concept of directions and character of destabilization of the cell genetic apparatus in the process of neoplastic transformation due to the MDR acquisition by cells.

A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1

Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all redox-active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1-reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1-dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS.

Studies of interaction of emodin and DNA in the presence of ethidium bromide by spectroscopic method

Emodin interacting with deoxyribonucleic acid (DNA) has been studied by different spectroscopic techniques, such as fluorescence, ultraviolet and visible (UV–vis), and fourier transform infared (FT-IR) spectroscopies, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA–EB system on addition of emodin shows that the fluorescence quenching of DNA–EB complex by emodin occurs. The binding constants of emodin with DNA in the presence of EB are 6.02 × 10 4, 9.20 × 10 4 and 1.17 × 10 5 L mol −1 at 20, 35 and 50 °C, respectively. FT-IR spectrum further suggests that both the phosphate groups and the bases of DNA react with emodin. The reaction of DNA with emodin in the presence of EB is affected by ionic strength and temperature. The values of melting temperature ( T m) of DNA–EB complex and emodin–DNA–EB complexes were determined, respectively. From the experiment evidences, the major binding mode of emodin with DNA should be the groove binding.

Molecular Analysis of the Interaction between the Hematopoietic Master Transcription Factors GATA-1 and PU.1

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Apically Sorted P2X7 Receptors Mediate Purinergic-Induced Pore Formation Preferentially in Apical Domains of the Plasma Membrane

Treatment of human epithelial cervical cells CaSki attached on filters with the P2X7-receptor (P2X7-R) agonist BzATP induced acute transient influx of calcium, most likely the result of P2X7-R channel activation, followed by slower sustained calcium influx. In cultures incubated in the presence of ethidium bromide (EB), BzATP induced slow and sustained influx of the dye with a time-course similar to the late slow calcium influx, suggesting P2X7-R pore formation. The acute and late calcium effects of BzATP were greater if the agonist was added to the luminal solution, facing the apical membrane of the cells. The EB effect of BzATP initially occurred in the apical membrane, while effects in the basolateral membrane were delayed and smaller. These results suggest that in polarized epithelial cells under steady-state conditions the P2X7-R is located in the apical membrane, and activation of the receptor induces formation of P2X7-R pores preferentially in the apical membrane.

Exploring writhe in supercoiled minicircle DNA

Using λ-Int recombination in E. coli, we have generated milligram quantities of supercoiledminicircle DNA. Intramolecular Int recombination was efficient down to lengths∼254 bp. When nicked and religated in the presence of ethidium bromide, 339 bp minicirclesadopted at least seven unique topoisomers that presumably correspond toΔLk rangingfrom 0 to −6, which we purified individually. We used these minicircles, with uniqueΔLk, to address the partition into twist and writhe as a function ofΔLk. Gel electrophoresis and atomic force microscopy revealedprogressively higher writhe conformations in the presence of 10 mMCaCl2 orMgCl2. From simplistic calculations of the bending and twisting energies, we predict the elasticfree energy of supercoiling for these minicircles to be lower than if the supercoiling waspartitioned mainly into twist. The predicted writhe corresponds closely with that which weobserved experimentally in the presence of divalent metal ions. However, in theabsence of divalent metal ions only limited writhe was observed, demonstratingthe importance of electrostatic effects on DNA structure, when the screening ofcharges on the DNA is weak. This study represents a unique insight into thesupercoiling of minicircle DNA, with implications for DNA structure in general.

DISTANT HYBRIDS AND THEIR VERIFICATION BY MEANS OF RAPD

In Poland, apart from Oriental lilies, Lilium longiflorum and its Asiatic hybrids, have become very popular. As a result of interspecific and intergroup hybridization, progeny plants may not always be hybrids. Embryos may be formed via apomixis and such seedlings contain the genetic material of the maternal form. Because apomixis can occur in Lilium regale, L. longiflorum, L. superbum and L. pumilum, confirmation that the obtained seedlings are hybrids is required. Studies identifying hybrids, based on DNA analysis began in the 1980’s. Molecular techniques, with the most popular one – RAPD, are used for identification. As a result of crossing Lilium longiflorum with ‘Connecticut King’, which is an Asiatic hybrid, progeny plants were obtained using embryo isolation method. The confirmation of their hybrid status was performed by RAPD, analysing DNA genotypes of two parental forms and six progeny plants. DNA was isolated from the leaves of young lilies growing in the greenhouse and 11 primers of 10-nucleotide sequences chosen at random, from LA pr 1 to LA pr 11 were used. Amplified markers by RAPD were separated on agar gel and visualized in UV light in the presence of ethidium bromide. Among evaluated genotypes five turned out to be hybrids and one had the same genotype as the maternal plant.

Polycations enhance emulsion-mediated in vitro and in vivo transfection

To enhance the in vitro and in vivo transfection activity of the cationic lipid emulsion (LE), three natural polycations, protamine sulfate (PS), poly- l-lysine and spermine, were selected as DNA condensing active agents. Formation of the LE/polycation/DNA ternary complexes was identified by using agarose gel retardation study. The structure of these complexes was characterized by measuring the complex size and the decrease of the DNA fluorescence in the presence of ethidium bromide (EtBr). By adding a polycation, the particle size of the complex decreased, and DNA in the complex became highly condensed and resistant to intercalation of EtBr. Among the polycations, PS yielded the most highly compacted ternary complex. In vitro and in vivo transfection activities of the complexes were determined using various cell lines and Balb/c mouse intravenously and intranasally, respectively. The transfection activity of the ternary complex increases by at least 2.5–5-fold in vitro cell culture system in the presence of 80% serum as well as in vivo mouse system, as compared with LE/DNA binary complexes. More importantly, after intravenous and intranasal administrations, the in vivo transfection efficiency of the LE/PS/DNA complex was ca. 30 and 50 times higher than that of the liposome (LP)/DNA complex in spleen and lung, respectively. On the other hand, cell toxicity of the ternary complex is lower than that of binary complex. Thus, we conclude that the pre-condensation of DNA with polycations can be a promising approach to further increase in vitro and in vivo transfection efficiency of cationic lipid emulsion.

Central role of Ifh1p–Fhl1p interaction in the synthesis of yeast ribosomal proteins

The 138 genes encoding the 79 ribosomal proteins (RPs) of Saccharomyces cerevisiae form the tightest cluster of coordinately regulated genes in nearly all transcriptome experiments. The basis for this observation remains unknown. We now provide evidence that two factors, Fhl1p and Ifh1p, are key players in the transcription of RP genes. Both are found at transcribing RP genes in vivo. Ifh1p, but not Fhl1p, leaves the RP genes when transcription is repressed. The occupancy of the RP genes by Ifh1p depends on its interaction with the phospho-peptide recognizing forkhead-associated domain of Fhl1p. Disruption of this interaction is severely deleterious to ribosome synthesis and cell growth. Loss of functional Fhl1p leads to cells that have only 20% the normal amount of RNA and that synthesize ribosomes at only 5-10% the normal rate. Homeostatic mechanisms within the cell respond by reducing the transcription of rRNA to match the output of RPs, and by reducing the global transcription of mRNA to match the capacity of the translational apparatus.

Stabilization of macromolecular chromatin complexes in mitotic chromosomes by light irradiation in the presence of ethidium bromide

A method was developed for stabilizing mitotic chromosomes. Light irradiation of permeabilized cells in a low concentration of ethidium bromide made chromatin resistant to high salt concentrations and decondensing buffer. This resistance was abolished by proteinase treatment, but not by DNase or RNase treatment. In photostabilized and extracted chromosomes, chromatin appeared as thick fibers with discrete high electron density regions. These stabilized structures might correspond to the higher-level structures (chromonemata) observed in native chromatin. Moreover, the electron density was higher in the centromeric regions than the chromosome arm material. Thus, the method allows chromatin substructures (chromonemata and centromeric heterochromatin) to be stabilized inside mitotic chromosomes.