Biomedical subjects

D Santamaría

Publications and source records attributed to D Santamaría.

7 recordsLinked to original sources

PubMed2000

Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins.

In Escherichia coli plasmids carrying two inversely oriented ColE1 origins, DNA replication initiates at only one of the two potential origins. The other silent origin acts as a replication fork barrier. Whether this barrier is permanent or simply a pausing site remains unknown. Here, we used a repeated primer extension assay to map in vivo, at the nucleotide level, the 5' end of the nascent strand where initiation and blockage of replication forks occurs. Initiation occurred primarily at the previously defined origin, however, an alternative initiation site was detected 17 bp upstream. At the barrier, the lagging strand also terminated at the main initiation site. Therefore, the 5' end of the nascent strand at the barrier was identical to that generated during initiation. This observation strongly suggests that blockage of the replication fork at the silent origin is not just a pausing site but permanent, and leads to a premature termination event.

Bacteriocin Plasmids↗

PubMed2000

Bi-directional replication and random termination.

Two-dimensional (2D) agarose gel electrophoresis was used to study termination of DNA replication in a shuttle vector, YRp7', when it replicated in Escherichia coli, Saccharomyces cerevisiae and Xenopus egg extracts. In E. coli, the 2D gel patterns obtained were consistent with uni-directional replication initiated at a specific site, the ColE1 origin. In consequence, termination also occurred precisely at the ColE1 origin. In Xenopus egg extracts, the particular shape of the bubble arc as well as the triangular smear detected to the left of the simple-Y pattern indicated random initiation and termination. In S.cerevisiae, initiation occurred at the ARS1 origin and replication proceeded in a bi-directional manner. However, termination did not always occur at a specific site 180 degrees across from the origin, but almost all along the south hemisphere of the plasmid. Inversion, deletion or replacement of DNA sequences located throughout this hemisphere did not eliminate random termination. Analysis of the replication intermediates of another yeast plasmid bearing a different origin, ARS305, also exhibited random termination. We propose that the random termination events observed in S.cerevisiae could be due to an asynchronous departure of both forks from the bi-directional origin in addition to differences in the rate of fork progression. These observations could be extended to all bi-directional origins.

Animals↗

PubMed2000

Effect of quinolinic acid on endogenous antioxidants in rat corpus striatum.

The response of endogenous antioxidants to the N-methyl-D-aspartate (NMDA) receptor agonist and excitotoxin, quinolinic acid (QUIN), was investigated in rat corpus striatum. Animals treated with QUIN (240 nmol/microl), were sacrificed at 120 min after a single intrastriatal injection to examine the alterations in the levels of both reduced (GSH) and oxidized (GSSG) glutathione, and the activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (Gpx). Changes in the rate of lipid peroxidation (LP) were also measured after exposure to different doses of QUIN (60, 120, 240 and 480 nmol/microl) as an index of oxidative stress. When compared to control, lipid peroxidation was increased at QUIN doses of 240 and 480 nmol/microl. Striatal levels of GSH and GSSG were decreased and increased, respectively, after QUIN injection; whereas GPx activity was unchanged. Cytosolic copper/zinc SOD (CuZn-SOD) activity decreased after treatment, while mitochondrial manganese SOD (Mn-SOD) was unchanged. The alterations observed on these antioxidant systems suggest that QUIN toxicity is mediated by specific mechanisms leading to oxidative stress.

Animals↗

PubMed1999

Nomega-nitro-L-arginine, a nitric oxide synthase inhibitor, antagonizes quinolinic acid-induced neurotoxicity and oxidative stress in rat striatal slices.

Nitric oxide (NO) is a potential contributor to neurotoxicity following overactivation of N-methyl-D-aspartate (NMDA) receptors. In this work we investigated the effect of Nomega-nitro-L-arginine (L-NARG 25, 50, or 100 microM), a selective inhibitor of nitric oxide synthase (NOS) -the synthetic enzyme of NO- on quinolinic acid (QUIN 100 microM)-induced neurotoxicity (measured as lactate dehydrogenase (LDH) leakage) in rat striatal slices. Oxidative stress was also measured both as lipid peroxidation and as the levels of reduced (GSH) and oxidized (GSSG) glutathione, in an effort to elucidate a possible participation of NO in the toxic mechanisms involved in NMDA receptor-mediated neuronal injury. The action of L-arginine (L-ARG 100 or 200 microM), a well-known NO precursor, was also tested on QUIN-induced neurotoxicity and oxidative stress. Results showed that QUIN produced significant changes in both cell damage (177%) and oxidative injury (203% in lipid peroxidation, 68% in GSH, and 123% in GSSG) as compared to control values. All these effects were antagonized by adding L-NARG to the incubation media, whereas L-ARG alone, or in combination with QUIN, significantly enhanced both lipid peroxidation and LDH leakage. Moreover, the protective effects of L-NARG on QUIN-induced lipid peroxidation were reversed by addition of an excess of L-ARG to the media. These findings indicate that NO is probably mediating the mechanism of neurotoxicity produced by QUIN, which may be of potential value to explain the molecular basis of neurodegenerative processes linked to QUIN-mediated NMDA receptor overactivation.

Animals↗

PubMed1998

DnaB helicase is unable to dissociate RNA-DNA hybrids. Its implication in the polar pausing of replication forks at ColE1 origins.

A series of plasmids were constructed containing two unidirectional ColE1 replication origins in either the same or opposite orientations and their replication mode was investigated using two-dimensional agarose gel electrophoresis. The results obtained showed that, in these plasmids, initiation of DNA replication occurred at only one of the two potential origins per replication round regardless of origins orientation. In those plasmids with inversely oriented origins, the silent origin act as a polar pausing site for the replication fork initiated at the other origin. The distance between origins (up to 5.8 kilobase pairs) affected neither the interference between them to initiate replication nor the pausing function of the silent origin. A deletion analysis indicated that the presence of a transcription promoter upstream of the origin was the only essential requirement for it to initiate replication as well as to account for its polar pausing function. Finally, in vitro helicase assays showed that Escherichia coli DnaB is able to melt DNA-DNA homoduplexes but is very inefficient to unwind RNA-DNA hybrids. Altogether, these observations strongly suggest that replication forks pause at silent ColE1 origins due to the inability of DnaB helicase, which leads the replication fork in vivo, to unwind RNA-DNA hybrids.

Autoradiography↗

PubMed1997

Cloning, sequencing and expression in MEL cells of a cDNA encoding the mouse ribosomal protein S5.

We describe the isolation and characterization of a cDNA encoding the mouse S5 ribosomal protein. It was isolated from a MEL (murine erythroleukemia) cell cDNA library by differential hybridization as a down regulated sequence during HMBA-induced differentiation. Northern series analysis showed that S5 mRNA expression is reduced 5-fold throughout the differentiation process. The mouse S5 mRNA is 760 bp long and encodes for a 204 amino acid protein with 94% homology with the human and rat S5.

Amino Acid Sequence↗

PubMed1997

Effects of N omega-nitro-L-arginine and L-arginine on quinolinic acid-induced lipid peroxidation.

The effects of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NARG), and a nitric oxide precursor, L-arginine (L-ARG), on the lipid peroxidation induced by quinolinic acid (QUIN, an NMDA receptor agonist), were both tested in synaptosomal fractions from whole rat brain. Baseline of lipid peroxidation was found at 2.43 +/- 0.24 fluorescence units/mg protein or 14.27 +/- 1.24 nmoles of TBARS/mg protein (100%). QUIN (100 microM)-induced lipid peroxidation in synaptosomes (256% and 166% vs. control, as measured by lipid fluorescent products and thiobarbituric acid-reactive substances, respectively) was inhibited by concentrations of 10, 40, 100, 200 and 400 microM of L-NARG (74%, 58%, 56%, 48% and 48% vs. quinolinate value, respectively). Coincubation of synaptosomes with QUIN plus L-ARG (100 microM), which alone resulted a potent pro-oxidant (277% vs. control), increased the lipoperoxidative effect induced by QUIN alone in 120% (290% vs. control). Synaptosomes simultaneously exposed to QUIN (100 microM) plus L-ARG (100 microM) plus L-NARG (200 microM) showed levels of lipid peroxidation similar to those of quinolinate alone. These findings suggest that nitric oxide may contribute to the oxidative damage induced in vitro by QUIN.

Animals↗