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R Eritja

Publications and source records attributed to R Eritja.

At least 19 recordsLinked to original sources
PubMed2006

Spectroscopic study of the interaction of actinomycin D with oligonucleotides carrying the central base sequences -XGCY- and -XGGCCY- using multivariate methods.

The interactions of actinomycin D (ACTD) with the oligonucleotides 5'-CAAAGCTTTG-3', 5'-CATGGCCATG-3' and 5'-TATGGCCATA-3' were investigated by means of acid-base titrations and mole-ratio and melting experiments monitored by molecular absorption and circular dichroism (CD) spectroscopies. For each experiment, CD and molecular absorption spectra were recorded at each point in the experiment, and later analyzed via appropriate multivariate data analysis methods. The study of the interactions between these oligonucleotides and ACTD at 25 degrees C showed the formation of an interaction complex with a stoichiometry of 1:1 (ACTD:duplex) and values for the log(formation constant) of 5.1+/-0.3, 6.4+/-0.2, and 5.6+/-0.2, respectively. An additional interaction complex at higher temperatures was also detected, which might be related to the single-stranded forms of the oligonucleotides.

Base Sequence↗
PubMed2006

First record and establishment of the mosquito Aedes albopictus in Spain.

The invasive mosquito Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) was detected for the first time in Spain, in Sant Cugat del Vallès, a city in the north-east of the country (41 degrees 28' N, 2 degrees 4' E, altitude 120 m), during August 2004. A male and one larva were collected in the backyard of a house and in a tree hole, respectively. Dense populations of adults and larvae were found in subsequent surveys, confirming the establishment of the species in the area. This is the first report of the establishment of this species in the Iberian Peninsula.

Aedes↗
PubMed2004

Synthesis, stability, and protonation studies of a self-complementary dodecamer containing the modified nucleoside 2'-deoxyzebularine.

The nucleoside 2'-deoxyzebularine (K) was incorporated into the self-complementary dodecamer 5'-CGTACGKGTACG-3' by solid-phase 2-cyanoethylphosphoramidite chemistry using dimethoxytrityl (DMT) as the 5'-hydroxyl protecting group. Standard synthesis cycles using trichloroacetic acid and short ammonia treatment (50 degrees C for 30 min) were found to be the optimal conditions to obtain the desired dodecamer with minimum acid and basic degradation of the acid- and base-sensitive 2-pyrimidinone residue. The protonation equilibria of the K nucleoside and of the dodecamer at 37 degrees C were studied by means of spectroscopically monitored titrations. For the K nucleoside, a pK(a) value of 3.13 +/- 0.09 was obtained. For the dodecamer, four acid-base species were found in the pH range 2-12, with pK(a) values of 9.60 +/- 0.07, 4.46 +/- 0.16, and 2.87 +/- 0.19. Melting experiments were carried out to confirm the proposed acid-base concentration profiles. Finally, kinetic experiments were also carried out at several pH values to evaluate the stability of the K nucleoside and of the dodecamer. An increased stability was shown by the K nucleoside when incorporated into the dodecamer. Multivariate methods based on both hard- and soft-modeling were applied for the analysis of spectroscopic data, allowing the estimation of concentration profiles and pure spectra.

Chromatography, High Pressure Liquid↗
PubMed2003

Resolution of parallel and antiparallel oligonucleotide triple helices formation and melting processes by multivariate curve resolution.

A procedure is described for the complete resolution of concentration profiles of oligonucleotide triplexes as a function of pH and temperature. The pH and temperature ranges at which triplexes are present and the relative concentrations of all the species involved in acid-base and conformational equilibria are successfully estimated from Multivariate Curve Resolution analysis of UV absorbance spectra recorded along acid-base titrations and melting experiments of single stranded, hairpin and their mixtures. The dependence of formation constants upon pH was successfully estimated. The hairpin h26 (5'-GAAGGAGGAGA-TTTT-TCTCCTCCTTC-3'), and the single stranded oligonucleotides s11CT (5'-CTTCCTCCTCT-3'), s11AG (5'-AGAGGAGGAAG-3') and s11TG (5'-TGTGGTGGTTG-3') were synthesized and their protonation and conformational equilibria were studied in detail. The procedure was shown to be especially useful for the study of triplexes with a low hypochromism upon formation.

Base Sequence↗
PubMed2001

Plasmid transcriptional repressor CopG oligomerises to render helical superstructures unbound and in complexes with oligonucleotides.

CopG is a 45 amino acid residue transcriptional repressor involved in the copy number control of the streptococcal plasmid pMV158. To do so, it binds to a DNA operator that contains a 13 bp pseudosymmetric DNA element. Binding of CopG to its operator results in repression, at the transcriptional level, of its own synthesis and that of the initiator of replication protein, RepB. Biochemical experiments have shown that CopG co-operatively associates to its target DNA at low protein:DNA ratios, completely protecting four helical turns on the same face of the double helix in both directions from the inverted repeat that constitutes the CopG primary target. This has been correlated with a CopG-mediated DNA bend of about 100 degrees. Here, we show that binding of CopG to DNA fragments containing the inverted repeat just at one end led to nucleation of the protein initiating from the inverted repeat. Nucleation extended to the entire fragment, with CopG-DNA contacts occurring on the same face of the DNA helix. The protein, the prototype for a family of homologous plasmid repressors, displays a homodimeric ribbon-helix-helix arrangement. It polymerises within the unbound crystal to render a continuous right-handed protein superhelix of homodimers, around which a bound double-stranded (ds) DNA could wrap. We have solved the crystal structure of CopG in complex with a 22 bp dsDNA oligonucleotide encompassing the cognate pseudosymmetric element. In the crystal, one protein tetramer binds at one face of the DNA with two parallel beta-ribbons inserted into the major groove. The DNA is bent about 50 degrees under compression of both major and minor grooves. A continuous right-handed complex helix made up mainly by protein-protein and some protein-DNA interactions is observed. The protein-protein interactions involve regions similar to those observed in the oligomerisation of the native crystals and those employed to set up the functional tetramer. A previously solved complex structure of the protein with a 19 bp dsDNA had unveiled a left-handed helical superstructure just made up by DNA interactions.

Bacterial Proteins↗
PubMed2001

The effect of amino groups on the stability of DNA duplexes and triplexes based on purines derived from inosine.

The effect of amino groups attached at positions 2 and 8 of the hypoxanthine moiety in the structure, reactivity and stability of DNA duplexes and triplexes is studied by means of quantum mechanical calculations, as well as extended molecular dynamics (MD) and thermodynamic integration (MD/TI) simulations. Theoretical estimates of the change in stability related to 2'-deoxyguanosine (G) --> 2'-deoxyinosine (I) --> 8-amino-2'-deoxyinosine (8AI) mutations have been experimentally verified, after synthesis of the corresponding compounds. An amino group placed at position 2 stabilizes the duplex, as expected, and surprisingly also the triplex. The presence of an amino group at position 8 of the hypoxanthine moiety stabilizes the triplex but, surprisingly, destabilizes the duplex. The subtle electronic redistribution occurring upon the introduction of an amino group on the purine seems to be responsible for this surprising behavior. Interesting 'universal base' properties are found for 8AI.

Base Sequence↗
PubMed2001

Protonation studies and multivariate curve resolution on oligodeoxynucleotides carrying the mutagenic base 2-aminopurine.

2-Aminopurine (P) is a mutagen causing A.T to G.C transitions in prokaryotic systems. To study the base-pairing schemes between P and cytosine (C) or thymine (T), two self-complementary dodecamers containing P paired with either C or T were synthesized, and their protonation equilibria were studied by acid-base titrations and melting experiments. The mismatches were incorporated into the self-complementary sequence d(CGCPCCGGXGCG), where X was C or T. Spectroscopic data obtained from molecular absorption, circular dichroism (CD), and molecular fluorescence spectroscopy were analyzed by a factor-analysis-based method, multivariate curve resolution based on the alternating least squares optimization procedure (MCR-ALS). This procedure allows determination of the number of acid-base species or conformations present in an acid-base or melting experiment and the resolution of the concentration profiles and pure spectra for each of them. Acid-base experiments have shown that at pH 7, 150 mM ionic strength, and 37 degrees C, both C and P are deprotonated. At pH near 4, the majority of species shows C protonated and P deprotonated. Finally, at pH values near 3, the majority of species shows both protonated C and P. These results are in agreement with NMR studies showing a wobble geometry for the P x C base pair and a Watson-Crick geometry for the P x T base pair at neutral pH. Melting experiments were carried out to confirm the proposed acid-base distribution profile. For the sequence including the P x T mismatch, only one transition was observed at neutral pH. However, for the sequence including the P x C mismatch, two transitions were detected by CD but only one by molecular absorption. This behavior agrees with that observed by other authors for oligonucleotides of similar sequence and suggests the following sequence of conformational changes during melting: duplex --> hairpin --> random coil.

2-Aminopurine↗
PubMed2001

Synthesis and properties of oligonucleotides containing 8-bromo-2'-deoxyguanosine.

The preparation of oligonucleotides containing 8-bromo-2'-deoxyguanosine is described. Substitution of G by 8-bromoguanine on an alternating CG decamer stabilizes the Z-form in such a way that the B-form was not observed. Melting temperatures showed that duplexes in which 8-bromo-2'-deoxyguanosine paired with natural bases were much less stable.

Chromatography↗
PubMed2001

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites.

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.

Base Sequence↗
PubMed2000

DNA-triplex stabilizing properties of 8-aminoguanine.

A DNA-triplex stabilizing purine (8-aminoguanine) is designed based on molecular modeling and synthesized. The substitution of guanine by 8-aminoguanine largely stabilizes the triplex both at neutral and acidic pH, as suggested by molecular dynamics and thermodynamic integration calculations, and demonstrated by melting experiments. NMR experiments confirm the triplex-stabilizing properties of 8-aminoguanine and demonstrate that few changes are found in the structure of the triplex due to the presence of this modified base.

Cytosine↗
PubMed2000

Synthesis and hybridization properties of DNA-PNA chimeras carrying 5-bromouracil and 5-methylcytosine.

The preparation of 5-bromouracil and 5-methylcytosine peptide nucleic acid (PNA) monomers is described. These PNA monomers were used for the preparation of several DNA-PNA chimeras and their hybridization properties are described. The substitution of cytosine by 5-methylcytosine in DNA-PNA chimeras increased duplex stability while substitution of thymine by 5-bromouracil maintained it. Moreover, binding of DNA-PNA chimeras to double-stranded DNA to form triple helices was studied. In contrast to DNA, the presence of 5-methylcytosine and 5-bromouracil in DNA-PNA chimeras destabilized triple helix.

5-Methylcytosine↗
PubMed1999

A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in Xenopus oocytes.

The activation of maturation-promoting factor (MPF) is required for G(2)/M progression in eukaryotic cells. Xenopus oocytes are arrested in G(2) and are induced to enter M phase of meiosis by progesterone stimulation. This process is known as meiotic maturation and requires the translation of specific maternal mRNAs stored in the oocytes. We have used an expression cloning strategy to functionally identify proteins involved in G(2)/M progression in Xenopus oocytes. Here we report the cloning of two novel cDNAs that when expressed in oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88% identical and have no significant homologies to other sequences in databases. These proteins, which we refer to as p33(ringo) (rapid inducer of G(2)/M progression in oocytes), induce very rapid MPF activation in cycloheximide-treated oocytes. Conversely, ablation of endogenous p33(ringo) mRNAs using antisense oligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33(ringo) is required for this process. We also show that p33(ringo) binds to and activates the kinase activity of p34(cdc2) but does not associate with p34(cdc2)/cyclin B complexes. Our results identify a novel p34(cdc2) binding and activating protein that regulates the G(2)/M transition during oocyte maturation.

Amino Acid Sequence↗
PubMed1999

"Action-at-a-distance" mutagenesis. 8-oxo-7, 8-dihydro-2'-deoxyguanosine causes base substitution errors at neighboring template sites when copied by DNA polymerase beta.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, favors a syn-conformation in DNA, enabling formation of stable 8-oxo-dG.A base mispairs resulting in G.C --> T.A transversion mutations. When human DNA polymerase (pol) beta was used to copy a short single-stranded gap containing a site-directed 8-oxo-dG lesion, incorporation of dAMP opposite 8-oxo-dG was slightly favored over dCMP depending on "downstream" sequence context. Unexpectedly, however, a significant increase in dCMP.A and dGMP.A mispairs was also observed at the "upstream" 3'-template site adjacent to the lesion. Errors at these undamaged template sites occurred in four sequence contexts with both gapped and primed single-stranded DNA templates, but not when pol alpha replaced pol beta. Error rates at sites adjacent to 8-oxo-dG were roughly 1% of the values opposite 8-oxo-dG, potentially generating tandem mutations during in vivo short-gap repair synthesis by pol beta. When 8-oxo-dG was replaced with 8-bromo-2'-deoxyguanosine, incorporation of dCMP was strongly favored by both enzymes, with no detectable misincorporation occurring at neighboring template sites.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Theoretical calculations, synthesis and base pairing properties of oligonucleotides containing 8-amino-2'-deoxyadenosine.

Theoretical calculations on double and triple helices containing 8-amino-2'-deoxyadenosine were made to analyze the possible differences in base pairing properties between 8-aminoadenine and adenine. These calculations indicate a strong preferential stabilization of the triplex over the duplex when adenine is replaced by 8-aminoadenine. In addition, a protected phosphoramidite derivative of 8-amino-2'-deoxyadenosine was prepared for the introduction of 8-aminoadenine into synthetic oligonucleotides using the phosphite-triester approach. DNA triple helical structures are normally observed at acidic pH. However, when oligonucleotides carrying 8-aminoadenine are used, very stable triple helical structures can be observed even at neutral pH. Biological applications of triple helices could benefit from the use of 8-aminoadenine derivatives.

Chromatography, High Pressure Liquid↗
PubMed1999

Environmental tobacco smoke interference in the assessment of the health impact of a municipal waste incinerator on children through urinary thioether assay.

The urinary elimination of thioethers urinary thioethers (UT) was studied in 83 schoolchildren living in two different areas of the city of Mataró, with special attention paid to the influence of a waste incinerator and of the smoking habits of their parents. The mean UT values were 8.79+/-3.23 and 7.52+/-3.23 mmol/mol creatinine in the area close to the incinerator (A1) and in the area far away from it (A2) respectively (statistically n.s.). Children exposed to environmental tobacco smoke (ETS) at home presented increased levels of UT (8.60+/-3.11 vs 5.93+/-3.22 mmol/mol creatinine; P=0.002). Considering the two exposures together (waste incinerator and ETS), no differences were found between the two areas studied (A1 and A2) in non-exposed (ETS) children, whereas slight differences were found when comparing highest ETS exposed children from the two areas (10. 18+/-2.70 vs 8.00+/-3.42 mmol/mol creatinine; P=0.04). Exposure to ETS could affect health more than pollutants from a waste incinerator and may interfere with non-selective assays such as urinary thioethers.

Air Pollutants↗