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Biomedical subjects

J Portugal

Publications and source records attributed to J Portugal.

At least 37 records · Page 2Linked to original sources
PubMed1992

Elsamicin A can convert the Z-form of poly[d(G-C)] and poly[(G-m5C)] back to B-form DNA.

The interaction of poly[(G-C)] and poly[d(G-m5C)] with the antitumor antibiotic elsamicin A, which binds to alternating guanine + cytosine tracts in DNA, has been studied under the B and Z conformations. Both the rate and the extent of the B-to-Z transition are diminished by the antibiotic, as inferred by spectroscopic methods under ionic conditions that otherwise favor the left-handed conformation of the polynucleotides. Moreover, elsamicin converts the Z-form DNA back to the B-form. The circular dichroism data indicate that elsamicin binds to poly[d(G-C)] and poly[d(G-m5C)] to form a right-handed bound elsamicin region(s). The transition can be followed by changes of the molar ellipticity at 250 nm, thus providing a convenient wavelength to monitor the Z-to-B conformational change of the polymers as elsamicin is added. The elsamicin A effect might be explained by a model in which the antibiotic binds preferently to a B-form DNA, playing a role as an allosteric effector on the equilibrium between the B and Z conformations, thus favoring the right-handed one.

Aminoglycosides↗
PubMed1992

Experimental and modelling studies on the DNA cleavage by elsamicin A.

The ability of elsamicin A, an antitumour antibiotic, to cleave DNA in the presence of ferrous iron and reducing agents, has been analysed using experimental and theoretical approaches. Experimentally, the antibiotic causes DNA breakage in the presence of ferrous ions and a reducing agent. The DNA-cleaving activity appears to be partially blocked by the action of superoxide dismutase and catalase. These results indicate that the elsamicin aglycone moiety (chartarin) can be involved in the production of free radicals. We have performed a broad theoretical study based in the quantum-mechanical framework, which allow us to determine the redox properties of elsamicin that lead to the generation of radical species. Our results clearly show that elsamicin acts as a true catalyst in the production of superoxide radicals. Moreover, it is suggested that the oxidation/reduction mechanism of the aglycone moiety of elsamicin (a lactone), leading to DNA breakage, is different from the mechanism followed by other well-known anti-cancer drugs, whose chromophore is a quinone.

Aminoglycosides↗
PubMed1992

Detection of elsamicin-DNA binding specificity by restriction enzyme cleavage.

The sequence specificity of elsamicin A, an anti-tumour antibiotic, binding to DNA was elucidated considering the inhibition of the rate of digestion of linearised pBR322 DNA by AatII, ClaI, EcoRI, HindIII and NruI restriction enzymes. Elsamicin A inhibits the rate of digestion by NruI (recognition sequence TCG/CGA) to a greater extent than it does for the other enzymes, thus evidencing the sequence-selective binding of elsamicin to CGC regions in DNA. Our results also show the important role of the neighbouring sequences in the elsamicin A-DNA interactions and their effects on the cleavage by restriction enzymes.

Aminoglycosides↗
PubMed1992

Interaction of trans-acting factors with the proximal promoter of the mouse alpha-fetoprotein gene.

1. The alpha-Fetoprotein (AFP) gene is expressed during fetal life, but not in adult cells. Also, the AFP gene is expressed in most hepatomas. 2. Using gel retardation (band-shift) assays under very stringent conditions we have compared the binding of trans-acting factors to the proximal enhancer (-202, +34) region of the AFP gene. 3. We have detected the presence of two retarded bands in experiments performed with adult rat hepatocytes and the Fa32 cell line (which does not produce AFP) but only one band is observed with the HepG2 cell line (which produces AFP) and fetal liver. 4. We relate the two retarded bands to a glucocorticoid response element and, tentatively, to the C/EBP trans-acting fractor.

Animals↗
PubMed1991

Map of chartreusin and elsamicin binding sites on DNA.

Three DNA restriction fragments designated tyrT, 102-mer and 70-mer, have been used as substrates for footprinting studies using DNase I in the presence of the structurally similar antibiotics chartreusin and elsamicin A. The sequence-selective binding sites of the antibiotics can be mapped in regions which are rich in guanine + cytosine. Chartreusin and elsamicin appear to recognize and bind preferentially to sequences containing a CpG step. Regions containing a TpG step also seem to be a good binding site. The binding of elsamicin to these sites appears to be more concentration-dependent. A comparative analysis is performed of the sizes and locations of the different binding sites, aimed to infer whether the different biological effects of chartreusin and elsamicin A can be correlated to differences in their sequence-selective binding to DNA.

Aminoglycosides↗
PubMed1991

Identification of sequence elements contributing to the intrinsic curvature of the mouse satellite DNA repeat.

In this paper, the contribution of different sequence elements to the intrisic curvature of the mouse satellite DNA repeat was investigated. This DNA fragment contains nineteen groups of three or more consecutive adenines which are only poorly phased with respect to the helical repeat. The mouse satellite DNA repeat shows a sinusoidal pattern of cleavage by the hydroxyl radical; the waves of reactivity are phased with respect to the A-tracts. Some interesting observations arise from a detailed analysis of these cleavage patterns: a) the maxima of hydroxyl radical cleavage are more periodically spaced along the DNA sequence than the A-tracts themselves. As a consequence, the position of each maximum with respect to the A-tract is variable; b) the sequence 5' TGGAATATG/AA 3' shows a sinusoidal pattern of hydroxyl radical cleavage. This sequence shows a retarded migration in polyacrylamide gels indicating that it is actually intrinsically curved. These results are discussed in view of the current models for DNA curvature.

Animals↗
PubMed1991

Cleavage of a four-way DNA junction by a restriction enzyme spanning the point of strand exchange.

The four-way DNA junction is believed to fold in the presence of metal ions into an X-shaped structure, in which there is pairwise coaxial stacking of helical arms. A restriction enzyme MboII has been used to probe this structure. A junction was constructed containing a recognition site for MboII in one helical arm, positioned such that stacking of arms would result in cleavage in a neighbouring arm. Strong cleavage was observed, at the sites expected on the basis of coaxial stacking. An additional cleavage was seen corresponding to the formation of an alternative stacking isomer, suggesting that the two isomeric forms are in dynamic equilibrium in solution.

Base Sequence↗
PubMed1990

Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone.

Base Sequence↗
PubMed1990

The tertiary structure of the four-way DNA junction affords protection against DNase I cleavage.

The accessibility of phosphodiester bonds in the DNA of four-way helical junctions has been probed with the nuclease DNase I. Regions of protection were observed on all four strands of the junctions, that tended to be longer on the strands that are exchanged between the coaxially stacked pairs of helices. The protected regions on the continuous strands of the stacked helices were not located exactly at the junction, but were displaced towards the 3' side of the strand. This is the region of backbone that becomes located in the major groove of the opposed helix in the non-crossed, right-handed structure for the junction, and might therefore be predicted to be protected against cleavage by an enzyme. However, the major grooves of the structure remain accessible to the much smaller probe dimethyl sulphate.

Alkylating Agents↗
PubMed1989

A model for the ability of drugs to induce enhanced DNase I cleavage.

A common property of sequence-selective DNA-binding drugs lies in their ability to induce an enhanced DNase I cleavage in regions surrounding their binding sites. A hypothetical model to explain the enhancements induced by drug binding to the minor-groove of DNA is presented. It involves the participation of three different single models: a mass action effect produced by the enzyme redistribution after drug binding; changes in the minor groove width size; and interactions between the enzyme and the drug, so increasing the cleavage in places located close to the binding site. The model is tested by using statistical data analysis. The hypothetical model might explain the experimental results better than any of the single models alone, but these models also appear to render significant results.

Base Sequence↗
PubMed1989

Footprinting analysis of sequence-specific DNA-drug interactions.

Footprinting is a technique widely used in the analysis of sequence-specificity of proteins and drugs which bind to DNA in an equilibrium manner. Footprinting studies combine the enzymatic or chemical cleavage of DNA with the analysis of the resulting products by sequencing gels, thus revealing the position and length of individual binding sites. This review compares the information that can be obtained by using different footprinting agents and the characteristics of DNA-drug interactions that can be inferred from footprinting results.

Base Sequence↗
PubMed1989

High resolution derivative denaturation profiles of DNA in the presence of copper(II) ions.

Derivative denaturation profiles of calf thymus DNA in the presence of copper(II) ions have been directly obtained from high resolution thermal denaturation profiles recorded in an isoabsorbance wavelength of the AT and GC hyperchromic spectra. The analysis of the very sensitive profiles provides further evidence that the melting temperature (Tm) of DNA decreases in the presence of stoichiometric ratio of copper(II) ions to nucleotide. Also, evidence is given of peculiar behaviour at higher temperatures where a new melting transition is observed. This phenomenon could be in line with the presence of bridging of DNA single strands by copper ions which are disrupted when the temperature is raised.

Animals↗
PubMed1989

Do Hoogsteen base pairs occur in DNA?

The importance of the Watson-Crick complementary base-pairing scheme has rather overshadowed alternative types of base pairs in DNA. One of these alternative base pairings, which is known as Hoogsteen pairing, is now receiving attention. Its presence in crystals of oligonucleotides bound to some antibiotics, and its possible existence in solution (and within long DNA fragments) remains to be unambiguously estimated. However, variability in DNA conformation appears to play an important biological role, and thus we should consider the presence of Hoogsteen base pairs as an interesting factor in inducing such changes.

Base Composition↗
PubMed1988

Diethyl pyrocarbonate can detect a modified DNA structure induced by the binding of quinoxaline antibiotics.

The reactivity of the 160 bp tyrT DNA fragment towards diethyl pyrocarbonate (DEPC) has been investigated in the presence of bis-intercalating quinoxaline antibiotics and the synthetic depsipeptide TANDEM. At moderate concentrations of each ligand, specific purine residues (mainly adenosines) exhibit enhanced reactivity towards the probe, and several sites of enhancement appear to be related to the sequence selectivity of drug binding. Further experiments were performed with echinomycin at pH 5.5 and 4.6 to facilitate the protonation of cytosine required for formation of Hoogsteen GC base pairs. No significant increase in reactivity was observed under these conditions. Additionally, no protection of deoxyguanosine residues from methylation by dimethyl sulphate was observed in the presence of echinomycin. We conclude that the structural anomaly giving rise to drug-dependent enhanced DEPC reaction is not simply the formation of Hoogsteen base pairs adjacent to antibiotic binding sites. Nor is it due to a general unwinding of the double helix, since we show that conditions which are supposed to unwind the helix lead to a uniform increase in purine reactivity, regardless of the surrounding nucleotide sequence.

Anti-Bacterial Agents↗
PubMed1988

Sequence-specific binding of luzopeptin to DNA.

We have examined the binding of luzopeptin, an antitumor antibiotic, to five DNA fragments of varying base composition. The drug forms a tight, possibly covalent, complex with the DNA causing a reduction in mobility on nondenaturing polyacrylamide gels and some smearing of the bands consistent with intramolecular cross-linking of DNA duplexes. DNAase I and micrococcal nuclease footprinting experiments suggest that the drug binds best to regions containing alternating A and T residues, although no consensus di- or trinucleotide sequence emerges. Binding to other sites is not excluded and at moderate ligand concentrations the DNA is almost totally protected from enzyme attack. Ligand-induced enhancement of DNAase I cleavage is observed at both AT and GC-rich regions. The sequence selectivity and characteristics of luzopeptin binding are quite different from those of echinomycin, a bifunctional intercalator of related structure.

Antibiotics, Antineoplastic↗
PubMed1988

Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.

DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions.

Benzimidazoles↗
PubMed1987

Hydroxyl radical footprinting of the sequence-selective binding of netropsin and distamycin to DNA.

Hydroxyl radicals, generated by allowing an iron (II).EDTA complex to react with hydrogen peroxide, have been employed to cleave the 160-base pair tyrT DNA fragment in the presence and absence of the minor groove-binding antibiotics netropsin and distamycin A. The control DNA cleavage pattern is practically independent of nucleotide sequence, which overcomes certain limitations of other footprinting techniques, so that additional information can be gained about the AT-rich sequence preference of the minor groove-binding ligands.

Base Composition↗