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Biomedical subjects

J Barbé

Publications and source records attributed to J Barbé.

At least 19 recordsLinked to original sources
PubMed2006

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

Biosensing Techniques↗
PubMed2003

LexA-binding sequences in Gram-positive and cyanobacteria are closely related.

The lexA gene of the cyanobacterium Anabaena sp. strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe. After over-expression in E. coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene. Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum. Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein. These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related.

Amino Acid Motifs↗
PubMed2001

Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines.

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties. This plant extract is being studied for treatment of viral diseases in animals and humans. Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property. Antimutagenesis was assayed against the mutagenic activity of m-phenylenediamine (m-PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium (S. typhimurium) YG1024, in different co-treatment approaches. This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m-PDA>2-AA>2-AF>9-AP>1-AP. Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis. Mutagenesis mediated by m-PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m-PDA and bacterial cells to the assay. Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic. In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay. Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m-PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds.

Amines↗
PubMed2001

Expression of the Pasteurella multocida ompH gene is negatively regulated by the Fur protein.

The fur gene of Pasteurella multocida has been cloned by complementation of an Escherichia coli fur mutant. The P. multocida fur gene, which encodes a predicted protein of 147 amino acids, displaying the highest identity (89%) with the same protein of Haemophilus influenzae, is negatively regulated by its own product. By construction of a P. multocida fur mutant, it has been demonstrated that the ompH gene, encoding a major structural protein of the outer membrane, presenting high antigenicity power, is negatively regulated by iron and glucose. Furthermore, wild-type and fur-defective cells of P. multocida show the same level of virulence.

Animals↗
PubMed2001

Virulence of Pasteurella multocida recA mutants.

In order to determine the role of the RecA protein in the virulence of Pasteurella multocida, a recA mutant was constructed and used in studies of virulence and competition in relation to wild-type strain. To achieve this, firstly, the recA gene was isolated and sequenced, showing an Escherichia coli-like SOS box and encoding a protein of 354 amino acids which has the closest identity with the Haemophilus influenzae RecA protein. Further, the recA mutant was constructed, by inactivating this gene by single recombination of a suicide plasmid containing an internal region of the P. multocida recA gene, and shown to be more sensitive to UV radiation than the parental strain. The P. multocida mutant was slightly attenuated in virulence, as indicated by the LD(50), the time of death of infected animals, and a failure to compete with the wild-type strain in mixed infections. Compared to the parent strain, the mutant had a similar growth rate but a longer lag phase. These data suggest that the diminished virulence of the recA mutant as well as its failure in competition were more a consequence of the long lag phase rather than a direct effect of the inactivation of the recA gene on genes involved in virulence.

Animals↗
PubMed2000

Plant activation of aromatic amines mediated by cytochromes P450 and flavin-containing monooxygenases.

To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s) and of the methimazole, a high-affinity substrate for FMOs, was studied. The efficacy of both inhibitors and of the methimazole was verified to find that they did partially inhibit the mutagenesis of both aromatic amines, activated with rat liver S9. Similarly, both inhibitors and methimazole did produce a significant decrease in 2-AF and m-PDA mutagenesis, when the activation system was S117, indicating that, similar to what occurs in mammalian systems, plant FMOs and cyt-P450s can metabolize aromatic amines to mutagenic product(s). However, the affinity of both FMOs and cyt-P450s of plant for 2-AF and m-PDA was different. Data obtained indicate that the activities of plant FMOs must be the main enzymatic system of m-PDA activation while, in 2-AF activation, plant cyt-P450s have the most relevant activities. In addition, peroxidases of the S117 system must contribute to 2-AF activation and some isoforms of FMOs and/or cyt-P450s of the S117 system, uninhibited by the inhibitors used, must be the responsible for a partial activation of m-PDA.

Amines↗
PubMed2000

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model.

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.

Adenosine Triphosphatases↗
PubMed2000

Analysis of the expression of the Rhodobacter sphaeroides lexA gene.

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN7GAACN7GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation.

Bacterial Proteins↗
PubMed2000

Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-9) from Escherichia coli in Spain.

A new CTX-M-type beta-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia coli strain. Despite the close identity that exists between the CTX-M-9 and Toho-2 beta-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.

Amino Acid Sequence↗
PubMed1999

A consensus sequence for the Rhodospirillaceae SOS operators.

The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis. Data obtained demonstrated that the GTTCN7GTAC and GAACN7GAAC motifs present upstream of the recA gene and the GTTCN7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction. Alignment of recA promoters of R. capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R. capsulatus and R. sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.

Adenosine Triphosphatases↗
PubMed1999

Regulation of divergent transcription from the uvrA-ssb promoters in Sinorhizobium meliloti.

The Sinorhizobium meliloti uvrA gene was isolated by complementation of a Rhodobacter sphaeroides UvrA- mutant. DNA sequencing of the region upstream of the S. meliloti uvrA gene reveals the presence of the ssb gene in the opposite transcriptional orientation. PCR-mediated mutagenesis demonstrated that expression of these two genes is inducible by DNA damage, and depends, in both cases, on the direct repeat GTTCN7GTTC (cited according to the direction of uvrA transcription). Comparison of the sequences of recA and uvrA promoters from different bacterial species of the alpha group of the Proteobacteria has identified the direct repeat GTTCYYKTTTTGTTC as the SOS box in this phylogenetic group.

Adenosine Triphosphatases↗
PubMed1998

Identification of the Rhodobacter sphaeroides SOS box.

Gel-mobility shift assays with crude cell extracts of Rhodobacter sphaeroides, which belongs to the alpha group of the proteobacteria, have shown that a protein binds to the promoter of its recA gene, resulting in two retardation bands. Analysis of the minimal region of the R. sphaeroides recA gene required for the formation of the DNA-protein complexes, revealed the presence of the motifs GTTCN7GATC and GAACN7GAAC, which are centred at positions -21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstrated that these two motifs are required for the formation of both DNA-protein complexes in vitro as well as for the DNA damage-mediated inducibility of the recA gene in vivo. Furthermore, the level of the recA gene expression in the constitutive mutants is the same as that achieved by the wild-type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN7GTTC is also present upstream of the R. sphaeroides uvrA promoter, which in vitro specifically binds to a protein and whose expression is DNA damage inducible. Mutagenesis of this motif abolishes both the binding of this protein to the uvrA promoter and the DNA damage-mediated expression of this gene. The fact that the recA and uvrA wild-type promoters compete with each other for the retardation band formation, but not with their mutant derivatives in any of these motifs, indicates that the same repressor binds to the operator of both genes. All these results lead us to propose the sequence GTTCN7GTTC as the SOS box of R. sphaeroides. This is the first SOS box known whose sequence is a direct repeat and not a palindrome.

Adenosine Triphosphatases↗
PubMed1998

Mutational analysis of the Rhizobium etli recA operator.

Based upon our earlier studies (A. Tapias, A. R. Fernández de Henestrosa, and J. Barbé, J. Bacteriol. 179:1573-1579, 1997) we hypothesized that the regulatory sequence of the Rhizobium etli recA gene was TTGN11CAA. However, further detailed analysis of the R. etli recA operator described in the present work suggests that it may in fact be GAACN7GTAC. This new conclusion is based upon PCR mutagenesis analysis carried out in the R. etli recA operator, which indicates that the GAAC and GTAC submotifs found in the sequence GAACN7GTAC are required for the maximal stimulation of in vivo transcription and in vitro DNA-protein complex formation. This DNA-protein complex is also detected when the GAACN7GTAC wild-type sequence is modified to obtain GAACN7GAAC, GTACN7GTAC, or GAACN7GTTC. The wild-type promoters of the Rhizobium meliloti and Agrobacterium tumefaciens recA genes, which also contain the GAACN7GTAC sequence, compete with the R. etli recA promoter for the DNA-protein complex formation but not with mutant derivatives in any of these motifs, indicating that the R. etli, R. meliloti, and A. tumefaciens recA genes present the same regulatory sequence.

Agrobacterium tumefaciens↗
PubMed1997

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli.

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

Amino Acid Sequence↗
PubMed1997

Importance of the galE gene on the virulence of Pasteurella multocida.

The galE gene of Pasteurella multocida has been isolated by complementing galE-defective mutants of Salmonella typhimurium with a plasmid library of this organism. The complete nucleotide sequence of the P. multocida galE gene consists of 1017 nucleotides, encoding a predicted polypeptide of 339 amino acids. The deduced amino acid sequence displayed the highest identity (85%) to the GalE protein of Haemophilus influenzae. However, the gene organization surrounding the galE locus was different from that of H. influenzae. A galE-defective mutant of P. multocida was obtained by replacement of the active galE gene by a copy inactivated in vitro. The resulting galE mutant was highly attenuated as seen in a biological test carried out in a mouse model.

Animals↗
PubMed1997

Expression of the Pseudomonas aeruginosa uvrA gene is constitutive.

By complementation of an Escherichia coli uvrA-defective mutant, the uvrA gene of Pseudomonas aeruginosa has been isolated from a genomic library. The nucleotide sequence of the promoter and the 5'-coding region of this gene have been determined. A P. aeruginosa SOS consensus region, which functions as a binding site for the LexA repressor molecule, was not present in the 629-bp upstream region of the cloned uvrA gene. Analysis of transcriptional fusions with the lacZ gene demonstrates that, contrary to what happens with its homologous gene of E. coli, the P. aeruginosa uvrA gene is not DNA damage-inducible. However, the UvrA protein must be functional in P. aeruginosa cells because an uvrA-defective mutant is extremely sensitive to UV radiation.

Adenosine Triphosphatases↗