Biomedical subjects

E Palecek

Publications and source records attributed to E Palecek.

At least 19 recordsLinked to original sources

PubMed2002

New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces.

Up to now, the development of the electrochemical DNA hybridization sensors relied on solid electrodes, on which both the hybridization and detection steps have been performed. Here we propose a new method in which the DNA hybridization is performed at commercially available magnetic beads and electrochemical detection on detection electrodes (DE). Due to minimum nonspecific DNA adsorption at the magnetic beads, very high specificity of the DNA hybridization is achieved. Optimum DE can be chosen only with respect to the given electrode process. It is shown that high sensitivity and specificity in the detection of relatively long target DNAs can be obtained (a) by using cathodic stripping voltammetry at mercury or solid mercury amalgam DEs for the determination of purine bases, released from DNA by acid treatment, and (b) by enzyme-linked immunoassay of target DNA modified by osmium tetroxide,2,2'-bipyridine (Os,bipy) at carbon DEs. Direct determination of Os,bipy at mercury and carbon electrodes is also possible.

Biosensing Techniques↗

PubMed2001

Determination of metallothionein at the femtomole level by constant current stripping chronopotentiometry.

Metallothionein (MT) from rabbit liver was analyzed by differential pulse polarography, cyclic voltammetry, square wave voltammetry, and chronopotentiometric stripping analysis (CPSA) with a hanging mercury drop electrode under various conditions. The highest sensitivity of the MT determination was obtained with CPSA which produced a well-developed peak H due to catalytic hydrogen evolution at highly negative potentials. The highest peak H was obtained in borate buffer close to pH 8.0. In this medium, subnanomolar concentrations of MT were detectable. In the adsorptive transfer stripping (medium exchange) experiments, determination of few femtomoles of MT in 5-microL aliquots of the analyte was possible. CPSA determination of MT in various tissues of carp (Cyprinus carpio) yielded values in agreement with the published data.

Animals↗

PubMed2001

Binding of p53 and its core domain to supercoiled DNA.

We have compared the binding of human full-length p53 protein (p53; expressed in bacteria and insects) and its isolated core domain (p53CD, amino acids 94-312; expressed in bacteria) to negatively supercoiled (sc) DNA using gel electrophoresis and immunoblotting. Significant differences were observed; p53CD produced a relatively small and continuous retardation of scDNA, in contrast to the ladder of distinct bands formed by p53 in agarose gels. The ladder produced by full-length protein expressed in bacteria (p53b) was similar to that observed earlier with protein expressed in insect cells (p53i). Competition between scDNAs and their linearized (lin) forms showed a preference for scDNAs by both p53 and p53CD, but the ratios characterizing the distribution of the protein between sc and lin pBluescript DNAs were substantially higher for p53 (sc/lin > 60 in p53b) than for p53CD (sc/lin approximately 4). Strong binding of p53 to scDNA lacking the p53 consensus sequence may represent a new p53-binding mode, which we tentatively denote supercoil-selective (SCS) binding. This binding requires both the C-terminal domain and the core domain. Targets of this binding may include: (a) DNA segments defined both by the nucleotide sequence and local topology, and/or (b) strand crossings and/or bending. The binding preference of p53CD for scDNA may be due to the known nonspecific binding to internal single-stranded regions in scDNA (absent in relaxed DNA molecules) and/or to SCS binding albeit with reduced affinity due to the absence of contributions from other p53 domains.

Baculoviridae↗

PubMed2000

Precise characterisation of monoclonal antibodies to the C-terminal region of p53 protein using the PEPSCAN ELISA technique and a new non-radioactive gel shift assay.

The development of human cancers is frequently associated with inactivation of the p53 tumour suppressor protein triggering cell cycle arrest or apoptosis in response to cellular stress. The p53 protein has been identified as a transcription factor with sequence-specific DNA binding properties. The DNA-binding activity is cryptic but can be modulated through the C-terminal region of the p53 protein by several different stimuli, including phosphorylation by casein kinase II (CKII), protein kinase C (PKC) or binding of the C-terminal monoclonal antibody PAb421. Monoclonal antibodies to the C-terminal region of p53 protein are able to activate the latent form of p53 and induce binding to DNA. To characterise such antibodies, we used a combination of the PEPSCAN ELISA procedure and a newly developed non-radioactive gel shift assay. Monoclonal antibodies from the Bp53 series displayed higher affinities for the human, rat and mouse p53 proteins than did the conventional antibody PAb421. In addition, these antibodies were able to activate the sequence-specific DNA binding functions in latent forms of p53 protein and, in contrast to PAb421, they were able to recognise both PKC phosphorylated and PKC non-phosphorylated forms of p53 protein. Our monoclonal antibodies recognising post-translationally modified target epitopes in the C-terminal region of p53 protein might assist the development of more effective molecules for p53-based cancer therapy.

Amino Acid Sequence↗

PubMed2000

Electrode potential-modulated cleavage of surface-confined DNA by hydroxyl radicals detected by an electrochemical biosensor.

Damage to DNA frequently involves interruption of DNA sugar-phosphate strands (strand breaks, sb). Under aerobic conditions, transition metal ions cause DNA damage through production of reactive oxygen species (frequently via Fenton-type reactions). Formation of sb in covalently closed supercoiled (sc) DNA can be detected using an electrochemical biosensor based on a scDNA-modified mercury electrode. By controlling the potential of the electrode, this technique can be employed in studies of redox reactions involved in formation of DNA strand breaks, and to detect species involved in these reactions. ScDNA anchored at HMDE was cleaved by catalytic amounts of iron/EDTA ions in the absence of chemical reductants when appropriate electrode potential (sufficiently negative to reduce [Fe(EDTA)]- to [Fe(EDTA)]2-) was applied. The process required oxygen or hydrogen peroxide. The extent of DNA damage increased with the shift of the electrode potential to negative values, displaying a sharp inflection point matching the potential of [Fe(EDTA)]2-/[Fe(EDTA)]- redox pair. In the absence of transition metal ions, significant DNA damage was observed at potentials sufficiently negative for reduction of dioxygen at the mercury electrode. This observation suggests cleavage of the surface-attached scDNA by radical intermediates of oxygen reduction at HMDE.

Biosensing Techniques↗

PubMed1999

DNA bending due to specific p53 and p53 core domain-DNA interactions visualized by electron microscopy.

We have used transmission electron microscopy to analyze the specificity and the extent of DNA bending upon binding of full-length wild-type human tumor suppressor protein p53 (p53) and the p53 core domain (p53CD) encoding amino acid residues 94-312, to linear double-stranded DNA bearing the consensus sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON). Both proteins interacted with high specificity and efficiency with the recognition sequence in the presence of 50 mM KCl at low temperature ( approximately 4 degrees C) while the p53CD also exhibits a strong and specific interaction at physiological temperature. Specific complex formation did not result in an apparent reduction of the DNA contour length. The interaction of p53 and the p53CD with p53CON induced a noticeable salt-dependent bending of the DNA axis. According to quantitative analysis with folded Gaussian distributions, the bending induced by p53 varied from approximately 40 degrees to 48 degrees upon decreasing of the KCl concentration from 50 mM to approximately 1 mM in the mounting buffer used for adsorption of the complexes to the carbon film surface. The p53CD bent DNA by 35-37 degrees for all salt concentrations used in the mounting buffer. The bending angle of the p53/DNA complex under low salt conditions showed a somewhat broader distribution (sigma approximately 39 degrees ) than at high salt concentration (sigma approximately 31 degrees ) or for p53CD (sigma approximately 24-27 degrees ). Together, these results demonstrate that the p53CD has a dominant role in complex formation and that the complexes formed both by p53 and p53CD under moderate salt conditions are similar. However, the dependence of the bending parameters on ambient conditions suggest that the segments flanking the p53CD contribute to complex formation as well. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed.

Base Sequence↗

PubMed1999

Effect of p53 protein redox states on binding to supercoiled and linear DNA.

The binding of p53 to its DNA consensus sequence is modulated by the redox state of the protein in vitro. We have shown previously that reduced wild-type p53 binds strongly to supercoiled DNA (scDNA) regardless of the presence or absence of p53CON. Here we compare the effects of oxidation of p53 by azodicarboxylic acid bis[dimethylamide] (diamide) and other agents on p53 binding to p53CON and to scDNA. Oxidation decreases the binding of p53 to scDNA; however, under conditions where binding to p53CON in a DNA fragment is completely abolished, some residual binding to scDNA is still observed. Increasing the concentration of oxidized p53 confers minimal changes in p53 binding to both scDNA and p53CON. Reduction of the oxidized protein by dithiothreitol neither restores its binding to DNA nor to p53CON in DNA fragments. In the presence of excess zinc ions, oxidation of p53 is, however, reversible. We conclude that the irreversibility of p53 oxidation is due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain accompanied by a conformational change of the p53 molecule after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein.

DNA↗

PubMed1999

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.

Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.

Antibodies↗

PubMed1999

Real-time monitoring of enzymatic cleavage of nucleic acids using a quartz crystal microbalance.

The use of quartz crystal microbalance (QCM) for monitoring in situ the enzymatic cleavage of surface-confined nucleic acids by nucleases is described. Such real-time monitoring of mass changes associated with the enzymatic digestion indicates that the activity and specificity of nucleases is preserved at the gold surface, and can be used for manipulating surface-confined DNAs and RNAs. These observations indicate great promise for using QCM for elucidating the interactions of nucleic acids with enzymes, and for enhancing the power of hybridization biosensors.

Crystallization↗

PubMed1999

Monoclonal antibody against DNA adducts with osmium structural probes.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

Animals↗

PubMed1998

Interactions of surface-confined DNA with acid-activated mitomycin C.

The anti-cancer drug mitomycin C (MC) was acid-activated and its interaction with single-stranded calf thymus DNA, immobilized at the surface of the hanging mercury drop electrode (DNA-modified HMDE) was studied by cyclic voltammetry. It was found that immersion of the DNA-modified electrode in a solution of acid-activated MC (at pH 3.9) for a short time (usually 1 min) at open current circuit, followed by transfer of the electrode in a neutral blank background electrolyte, resulted in a decrease of the anodic peak G (due to guanine residues in DNA) and in the formation of a reversible couple at approx. -0.44 V. The potential of the cathodic peak was approx. 50 mV more negative than the cathodic peak of the acid-activated MC obtained under the same conditions in the absence of DNA. No changes of peak G occurred and only a very small cathodic peak appeared if the DNA-modified electrode was immersed in an MC solution not exposed to acid pH. On the basis of these results and additional experiments, including dependence on concentration, time and pH during the interaction of MC with DNA at the electrode surface, we concluded that acid-activated MC is covalently bound to guanine residues in DNA immobilized at the electrode surface and that the quinone group in the DNA-MC adduct is reversibly reduced at the electrode.

Animals↗

PubMed1998

Electrochemical biosensors for DNA hybridization and DNA damage.

Recent trends in the development of DNA biosensors for nucleotide sequence-specific DNA hybridization and for the detection of the DNA damage are briefly reviewed. Changes in the redox signals of base residues in DNA immobilized at the surface of carbon or mercury electrodes can be used as a sign of the damage of DNA bases. Some compounds interacting with DNA can produce their own redox signals on binding to DNA. Covalently closed circular (usually supercoiled) DNA attached to the electrode surface can be used for a sensitive detection of a single break of the DNA sugar-phosphate backbone and for detection of agents cleaving the DNA backbone such as hydroxyl radicals, ionizing radiation, nucleases, etc. Using the peptide nucleic acid in the biosensor recognition layer greatly increased the specificity of the DNA hybridization biosensor making it possible to detect point mutations (single-base mismatches) in DNA.

Animals↗

PubMed1998

Analysis of a curved DNA constructed from alternating dAn:dTn-tracts in linear and supercoiled form by high resolution chemical probing.

Complex of osmium tetroxide and bipyridine (Os,bipy), KMnO4, and diethyl pyrocarbonate (DEPC) were used to probe curved DNA at single nucleotide resolution. The DNA was constructed from repeated dAn:dTn-blocks with dATATA and dAGAGA interblock sequences. The DNA was probed in the linear and supercoiled form at various salt concentrations. While all purines were available for DEPC attack, the thymines within the blocks were resistant to chemical probing by KMnO4 and Os,bipy. Only the 3'-flanking dTs were available for modification. The thymines within dTC and dTA sequences showed modification indicating that these thymines display an unstacked structure allowing both probes to attack. Under destabilizing conditions, at low ionic strength and superhelical stress, considerable unstacking was observed. We found experimental indications that under these destabilizing conditions unpaired regions might appear, probably within the dATATA sequence.

DNA, Superhelical↗

PubMed1998

Two superhelix density-dependent DNA transitions detected by changes in DNA adsorption/desorption behavior.

The adsorption behavior of covalently closed circular plasmid DNA at the mercury/water interface was studied by means of AC impedance measurements. The dependence of the differential capacitance (C) of the electrode double layer on the potential (E) was measured in the presence of adsorbed DNA. It was found that the C-E curves of supercoiled DNA at native and highly negative superhelix densities (sigma), relaxed covalently closed circular DNA, and nicked DNA differed from each other. A detailed study of topoisomer distributions ranging from -sigma of 0 to 0.11 revealed two supercoiling-dependent transitions, at about -sigma = 0.04 (transition TI) and 0.07 (transition TII). Transition TI was detected by measuring the height of the adsorption/desorption peak 1 (at about -1.2 V against the saturated calomel electrode) and the decrease of capacitance (DeltaC) at -0.35 V. This transition may be due to a sudden change in the ability of the DNA to respond to the alternating voltage, probably caused by changes in the DNA tertiary and/or secondary structure. Transition TII was detected by measuring peak 3* (at about -1.3 V), which was absent in topoisomers with -sigma less than 0.05. This transition is due to changes in the DNA adsorption/desorption behavior related to increased accessibility of bases at elevated negative superhelix density. Opening of the duplex at highly negative superhelix density was also detected by the single-strand selective probe of DNA structure, osmium tetroxide, 2, 2'-bipyridine. Our results suggest that electrochemical techniques provide sensitive experimental analysis of changes in DNA structure.

Adsorption↗

PubMed1997

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.

Adsorption↗

PubMed1997

Tumor suppressor protein p53 binds preferentially to supercoiled DNA.

Wild type human tumor suppressor protein p53 (expressed in insect cells) binds strongly to negatively supercoiled (sc) plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of scDNA in agarose gels and imaging by scanning force microscopy (SFM). The binding occurs both in the presence and absence of the p53 consensus sequence. At relatively low p53/DNA ratios, binding of p53 to scDNA results in the appearance of several retarded DNA bands on the gels, similar to a conventional topoisomer ladder generated enzymatically. However, after removal of p53 by deproteination, the original mobility of the scDNA is recovered, indicating that the reduction of torsional stress accompanying p53 binding does not reflect changes in linking number. In DNA samples partially relaxed by topoisomerase I p53 binds preferentially to the scDNA molecules with the largest negative superhelix density. SFM imaging of the p53/scDNA complex reveals a partial or total relaxation of the compact scDNA, the degree of which increases with the number of bound p53 molecules. Competition assays with linear DNA reveal a preference of p53 for scDNA. In addition, scDNA induces dissociation of p53 from a preformed complex with a DNA fragment (474 bp) containing the consensus sequence. We conclude that the affinity of p53 for negatively supercoiled DNA is greater than that for the consensus sequence in linear fragments. However, thermally denatured linearized plasmid DNA is efficient in competing for the binding of p53 to scDNA, although the first retarded band (presumed to contain one bound p53 molecule) is retained in the case of the plasmid containing the consensus sequence. Thus, it appears that interactions involving both the core domain and the C-terminal domain regulate the binding of p53 to scDNA. The above results are not restricted to human p53; the wild type rat p53 protein also results in the retardation of scDNA on agarose gels. The biological implications of the novel DNA binding activities of p53 are discussed.

Animals↗

PubMed1996

Effect of flanking sequences on the right- to left-handed transition of a (dA-dT)n tract in supercoiled DNA.

Alternating (dA-dT)n sequences in supercoiled DNA may undergo a transition to a left-handed conformation in the presence of Ni2+ ions and high NaCl concentration (Nejedlý, K., Klysik, J. and Palecek, E., FEBS Lett. 243, 313-317 (1989)). In this work we have found that ionic conditions necessary for the B-to-Z transition are strongly dependent on the sequences flanking the (dA-dT)n tract. In particular, the presence of 5'-homopyrimidine (C3) and 3'-homopurine (G4) blocks adjacent to the tract were found to facilitate the transition to the left-handed form. Within a constant sequence context it was found that the ionic strength required to promote the transition was inversely proportional to the length of the (dA-dT)n sequence.

Base Sequence↗

PubMed1995

Intramolecular TAT triplex in (dA)58.(dT)58. influence of ions.

Supercoil-stabilized intramolecular triplexes have been described under various conditions in different polypurine.polypyrimidine sequences such as (dG)n.(dC)n and mixed sequences including d(GA)n.d(CT)n while information about the triplexes in (dA)n.(dT)n is scarce. Using osmium tetroxide complexes and diethyl pyrocarbonate as structural probes, we show a pyrimidine.purine.pyrimidine (TAT) triplex in (dA)58.(dT)58 sequence in a supercoiled plasmid pE19. Strong modification of approximately six central thymines and approximately six T's at the 3'-end of the (dT)58 stretch as well as the DEPC modification of the 5'-half of the (dA)58 strand suggested the prevalence of the H-y3 triplex conformer. At native superhelix density, optimum conditions for the triplex formation were close to 1 mM MgCl2, pH 8.5. At room temperature and MgCl2 concentrations below 0.5 and above 5 mM, almost no triplex was formed. It is suggested that the absence of the triplex at higher MgCl2 concentrations is due to the stabilization of the duplex by Mg2+ ions which prevents the duplex opening necessary for the triplex formation. At higher temperatures, favorable for duplex opening (e.g. 55 degrees C), the TAT triplex is formed even in the presence of 10 mM MgCl2. Among Ca2+, Sr2+, Ba2+, Cd2+, Zn2+ and Ni2+, only Ca2+ and Sr2+ yielded a modification pattern similar to that obtained with Mg2+; the modification pattern produced in the presence of Sr2+ was, however, much less intense. In the presence of 1 mM MgCl2, a decrease in pH from 8.5 to 7.7 resulted in a strong decrease of the triplex content. At highly negative superhelix density, the conditions for triplex formation were less stringent, and the triplex was observed even in the absence of MgCl2.

Base Composition↗