Biomedical subjects
N Pastor
Publications and source records attributed to N Pastor.
[Umbilicated papules on genitals in two siblings].
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Pyoderma gangrenosum treated with anti-TNF alpha therapy (etanercept).
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Induction of genotoxic and cytotoxic damage by aclarubicin, a dual topoisomerase inhibitor.
The anthracycline aclarubicin (ACLA) is an intercalative antibiotic and antineoplastic agent that efficiently binds to DNA, leading to a secondary inhibition of the catalytic activity of topoisomerase II (topo II) on DNA. Besides this activity, ACLA has been reported to exert a concomitant poisoning effect on topo I, in a fashion similar to that of the antitumor drug camptothecin and its derivatives. As a consequence of this dual (topo II catalytic inhibiting/topo I poisoning) activity of ACLA, the picture is somewhat confusing with regards to DNA damage and cytotoxicity. We studied the capacity of ACLA to induce catalytic inhibition of topo II as well as cytotoxic effects and DNA damage in cultured Chinese hamster V79 cells and their radiosensitive counterparts irs-2. The ultimate purpose was to find out whether differences could be observed between the two cell lines in their response to ACLA, as has been widely reported for radiosensitive cells treated with topo poisons. Our results seem to agree with the view that the radiosensitive irs-2 cells appear as hypersensitive ACLA as compared with radiation repair-proficient V79 cells. The recovery after ACLA treatment was also followed-up, and the irs-2 mutant was found to be less proficient than V79 to repair DNA strand breaks induced by ACLA.
Erythema multiforme due to methotrexate reproduced with patch test.
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Allergic contact gingivitis due to eugenol present in a restorative dental material.
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DNA strand breaks induced by the anti-topoisomerase II bis-dioxopiperazine ICRF-193.
The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.
Testing the SCE mechanism with non-poisoning topoisomerase II inhibitors.
There are controversial theoretical models about a possible involvement of DNA topoisomerase II (topo II) in the molecular mechanism of sister chromatid exchanges (SCEs). In order to clarify the role of this enzyme, if any, in such recombinational event, CHO parental AA8 and mutant EM9 cells, which shows and extremely high baseline frequency of SCE, have been treated with different doses of the non-poisoning topoisomerase inhibitors, ICRF-193 and bufalin. The frequencies of SCEs after the treatments have been determined and the inhibitory effect of these compounds has been assessed using a topo II activity assay. The results indicate that ICRF-193 and bufalin effectively inhibit topo II activity in AA8 and EM9 cell lines. ICRF-193 induced a moderate increase in the frequency of SCEs in both types of cells, while bufalin did not modify the level of SCEs in any of them. The results are discussed taking into account the apparently unlike mechanisms of inhibition of topo II by ICRF-193 and bufalin.
Assessment of genotoxic damage by the comet assay in white storks (Ciconia ciconia) after the Doñana Ecological Disaster.
Single cell gel electrophoresis, the so-called "Comet" assay, was performed as a genotoxicity test in white storks sampled in an area heavily contaminated after the ecological disaster in south western Spain. This disaster occurred as a consequence of a massive toxic spillage of acid waste rich in heavy metals that impacted on the Doñana National Park. The importance of this protected area as a breeding and wintering site for many endangered bird species makes this analysis of DNA damage of special interest. Our results clearly show that white storks born in the contaminated area 1 year after the toxic spill bear a high burden of genetic damage as compared with control individuals. The possible implications for future survival as well as reproductive rate are discussed.
Ionizing radiation damage repair: a role for topoisomerases?
In parallel with the developing field of DNA topoisomerase poisons in tumor chemotherapy, the basic features of these nuclear enzymes have been unfolded. The role of topoisomerases in fundamental processes involving DNA metabolism has been shown to outpace by far the initial expectations. While DNA topoisomerases are involved in relaxation of chromatin to relieve tension during DNA replication and transcription, as well as for recombinational processes and chromosome segregation and condensation, the possible role, either direct or indirect, of these enzymes in DNA repair is still a matter of discussion. In this survey the possible relationship of topoisomerases with the repair of ionizing radiation damage in mammalian cells is considered, on the basis of attractive 'clues' and in the light of a number of observations.
DNA topoisomerases in cancer chemotherapy: basic and applied aspects.
These conserved nuclear enzymes catalyse a variety of interconversions of DNA which take place between topological isoforms of the molecule, through transient cleavage, strand passing and re-ligation. In this context the DNA topoisomerases have been generally considered as good candidates to play a role in the enzymatic repair processes going on in the cell after DNA damage inflicted by either physical or chemical agents. It has been suggested that this role could be direct, with the actual participation of topoisomerases in the molecular mechanism of lesion repair, as for example in the excision of base damage or DNA strand break repair. Alternatively, it could be indirectly responsible for the preparative steps in the relaxation of chromatin in order to allow repair enzymes to gain access to damaged DNA.
DNA damage in birds after the mining waste spill in southwestern Spain: a Comet assay evaluation.
In April 1998, an ecological disaster resulting from a massive toxic spill of mining acid waste rich in heavy metals posed a serious threat to the Doñana National Park in southwestern Spain. This especially important protected area is the nesting and breeding site for many endangered bird species; white storks (Ciconia ciconia) and black kites (Milvus migrans) are considered the more representative. The suitability of the Comet assay as a biomarker for genotoxic analysis in environmental biomonitoring has been recently validated in studies using different sentinel organisms such as fish, amphibians, rodents, or mollusks. Birds preying on a variety of invertebrate and vertebrate species in the marshlands are appropriate for evaluating the potential deleterious effects of the toxic spill on wildlife of the Dofiana area. Our study on wetland birds high on the aquatic trophic chain sampled within a few months after the toxic spill in the area around Doñana National Park has shown the accumulation of heavy metals. Fourteen months after the mine waste spill, blood samples from white storks and kites collected in the neighborhood of the park and from control birds at reference areas for comparison were examined by fluorescence image analysis after lymphocyte isolation, and by subsequent alkaline single-cell gel (SCG) electrophoresis, known as the Comet assay. Our results indicate that the exposed birds had a significantly increased level of genotoxic damage compared with control animals from noncontaminated locations.
A detailed interpretation of OH radical footprints in a TBP-DNA complex reveals the role of dynamics in the mechanism of sequence-specific binding.
The hydroxyl radical footprint of the TATA-binding protein (TBP) bound to the high-affinity sequence TATAAAAG of the adenovirus 2 major late promoter has been quantitatively compared to a 2 ns molecular dynamics simulation of the complex in aqueous solution at room temperature using the CHARMM23 potential. The nucleotide-by-nucleotide analysis of the TBP-TATA hydroxyl radical footprint correlates with the solvent-accessible surface calculated from the dynamics simulation. The results suggest that local reactivity towards OH radicals results from the interplay between the local DNA geometry imposed by TBP binding, and the dynamics of the side-chains contacting the sugar hydrogen atoms. Analysis of the dynamics suggests that, over time, TBP forms stable interactions with the sugar-phosphate backbone through multiple contacts to different partners. This mechanism results in an enthalpic advantage to complex formation at a low entropic cost.
Binding mechanisms of TATA box-binding proteins: DNA kinking is stabilized by specific hydrogen bonds.
One of the common mechanisms of DNA bending by minor groove-binding proteins is the insertion of protein side chains between basepair steps, exemplified in TBP (TATA box-binding protein)/DNA complexes. At the central basepair step of the TATA box TBP produces a noticeable decrease in twist and an increase in roll, while engaging in hydrogen bonds with the bases and sugars. This suggests a mechanism for the stabilization of DNA kinks that was explored here with ab initio quantum mechanical calculations and molecular dynamics/potential of mean force calculations. The hydrogen bonds are found to contribute the energy necessary to drive the conformational transition at the central basepair step. The Asn, Thr, and Gly residues involved in hydrogen bonding to the DNA bases and sugar oxygens form a relatively rigid motif in TBP. The interaction of this motif with DNA is found to be responsible for inducing the untwisting and rolling of the central basepair step. Notably, direct readout is shown not to be capable of discriminating between AA and AT steps, as the strength of the hydrogen bonds between TBP and the DNA are the same for both sequences. Rather, the calculated free energy cost for an equivalent conformational transition is found to be sequence-dependent, and is calculated to be higher for AA steps than for AT steps.
TIT for TAT: the properties of inosine and adenosine in TATA box DNA.
The sequence dependent conformation, flexibility and hydration properties of DNA molecules constitute selectivity determinants in the formation of protein-DNA complexes. TATA boxes in which AT basepairs (bp) have been substituted by IC bp (TITI box) allow for probing these selectivity determinants for the complexation with the TATA box-binding protein (TBP) with different sequences but identical chemical surfaces. The reference promoter Adenovirus 2 Major Late Promoter (mlp) is formed by the apposition of two sequences with very different dynamic properties: an alternating TATA sequence and an A-tract. For a comparative study, we carried out molecular dynamics simulations of two DNA oligomers, one containing the mlp sequence (2 ns), and the other an analog where AT basepairs were substituted by IC basepairs (1 ns). The simulations, carried out with explicit solvent and counterinons, yield straight purine tracts, the A-tract being stiffer than the I-tract, an alternating structure for the YRYR tracts, and hydration patterns that differ between the purine tracts and the alternating sequence tracts. A detailed analysis of the proposed interactions responsible for the stiffness of the purine tracts indicates that the stacking between the bases bears the strongest correlation to stiffness. The hydration properties of the minor groove in the two oligomers are distinctly different. Such differences are likely to be responsible for the stronger binding of TBP to mlp over the inosine-substituted variant. The calculations were made possible by the development, described here, of a new set of forcefield parameters for inosine that complement the published CHARMM all-hydrogen nucleic acid parametrization.
Topoisomerase activities and levels in irradiated Chinese hamster AA8 cells and in its radiosensitive mutant EM9.
PURPOSE: To investigate possible variations in topoisomerase (topo) I and II activities and levels after X-ray treatment in the radiation repair proficient AA8 Chinese hamster cell line for comparison with the radiation sensitive mutant EM9. MATERIALS AND METHODS: AA8 and EM9 cells were irradiated with 5 Gy of X-rays and the activities of topoisomerases I and II in nuclear extracts were studied. Immunological detection of both topoisomerases was carried out in order to detect any changes in the expression of these enzymes as a consequence of irradiation. RESULTS: Topoisomerase activities and levels in irradiated EM9 cells were the same as in control non-irradiated cells. In fact, both topo I and topo II activities clearly increased shortly after irradiation in the parental AA8 cells, with a more rapid increase for topo I than for topo II. In the AA8 cells, an increased level of topo I detectable immunologically was only observed at a later time (1 h) after irradiation, while no similar change was detectable for topo II. CONCLUSIONS: While this hypothesis needs further testing, an attractive idea is that DNA topoisomerases might be involved in the cellular response to radiation damage, either through a direct participation in repair mechanisms or indirectly.
Selective binding of the TATA box-binding protein to the TATA box-containing promoter: analysis of structural and energetic factors.
We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding -NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.
Progressive DNA bending is made possible by gradual changes in the torsion angle of the glycosyl bond.
Structural comparisons have led to the suggestion that the conformational rearrangement that would be required to change A-DNA into the TA-DNA form of DNA observed in the complex with the TATA box binding protein (TBP) could be completed by modifying only the value of the glycosyl bond chi by approximately 45 degrees. The lack of a high number of crystal structures of this type makes it difficult to conclude whether a smooth transition from A-DNA to TA-DNA can occur without disrupting at any point either the Watson-Crick base pairing or the A-DNA conformation of the backbone. To explore the possibility of such a smooth transition, constrained molecular dynamics simulations were carried out for the double-stranded dodecamer d(GGTATATAAAAC), in which a transition from A-DNA to TA-DNA was induced by modifying only the chi angle values. The results demonstrate the feasibility of a continuous path in the A-DNA to TA-DNA transition. Varying extents of DNA curvature are also attainable, by maintaining the A-DNA backbone structure and Watson-Crick hydrogen bonding while changing the chi angle value smoothly from that in A-DNA to one corresponding to B-DNA.