Novel blaROB-1-Bearing Plasmid Conferring Resistance to β-Lactams in Haemophilus parasuis Isolates from Healthy Weaning Pigs

Haemophilus parasuis, the causative agent of Glässer''s disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the bla_ROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer''s disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.     

Novel S-ribosylhomocysteine analogues as potential inhibitors of LuxS enzyme

Selective cross-coupling of the protected 6-fluoro-6-iodo-alpha-D-ribo-hex-5-enofuranose with 2 equivalents of 4-ethoxy-4-oxobutylzinc bromide in the presence of Pd[P(Ph)(3)](4) followed by deprotections gave methyl 5,6,7,8,9-pentadeoxy-6-fluoro-alpha/beta-D-ribo-dec-5(Z)-enofuranuronate; a S-ribosylhomocysteine analogue with the sulfur and carbon-5 atoms replaced by the fluoro(vinyl) unit.     

À la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XylR to non-natural effectors

To investigate the activation mechanism of the enhancer-binding protein XylR encoded by the TOL plasmid of Pseudomonas putida mt-2, a combinatorial library was generated composed of shuffled N-terminal A domains of the homologous regulators DmpR, XylR and TbuT, reassembled within the XylR structure. When the library was screened in vivo for responsiveness to non-effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely different distribution of electronegative groups (e.g. nitrotoluenes), protein variants were found that displayed an expanded inducer range including the new effectors. Although the phenotypes endowed with the corresponding changes were largely similar, the modifications involved different sites within the A domain. The positions of the mutations within a structural model of the A domain suggest that expansion of the inducer profile can be brought about not only by changes in the effector pocket of the protein but also by unlocking steps of the signal transmission mechanism that follows effector binding. These results provide a rationale for evolving in vitro regulators à la carte that are responsive to predetermined, natural or xenobiotic chemical species.     

Immunocytochemical finding of the amidating enzymes in mouse pancreatic A-, B-, and D-cells: a comparison with human and rat.

alpha-Amidation is catalyzed by two enzymatic activities, peptidyl-glycine alpha-hydroxylating mono-oxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), denoted collectively as peptidyl-glycine alpha-amidating mono-oxygenase (PAM), which also may include transmembrane and cytoplasmic domains. PAM is present in mammalian pancreas, where it appears to be abundant in the perinatal period. Nevertheless, there is no agreement on the cell type(s) that produces PAM or even on its presence in adults. In the present study we found PAM (PHM and cytoplasmic domain) immunoreactivity (IR) in A-, B-, and D-cells of adult mouse pancreas. In contrast to previous reports, PAM IR was found in B-cells of human and rat. Most of the B/D-cells were PAM immunoreactive, although with variable intensity, whereas less than half of A-cells displayed IR. Immunocytochemistry and Western blotting suggested the existence of different PAM molecules. Differences in the cellular distribution of IR for PAM domains were also observed. Whereas PHM-IR was extended throughout the cytoplasm in the three cell types, presumably in the secretory granules, IR for the cytoplasmic domain in A/D-cells was restricted to a juxtanuclear region, perhaps indicating its cleavage in Golgi areas. Although glucagon, insulin, and somatostatin are non-amidated, amidated peptides (glucagon-like peptide 1, adrenomedullin, proadrenomedullin N-terminal 20 peptide) were found in the three cell types.     

Analysis of foot-and-mouth disease virus-neutralizing idiotypes from immune bovine and swine with anti-murine idiotype antibody probes

Rabbit anti-idiotypic antibodies (a-IdAb) induced by foot-and-mouth disease virus (FMDV) neutralizing mAb were used as probes to identify anti-FMDV Id in immune serum from bovine and swine. In a competitive RIA, at least two of the a-IdAb exhibited a dose-dependent capacity to compete with labeled virus for anti-FMDV antibodies from a convalescent bovine serum. These a-IdAb were immobilized on activated Sepharose and used to isolate anti-viral Id from bovine, swine, and murine FMDV immune sera. Both the bovine and swine antibodies recovered from the a-IdAb/Sepharose columns reacted with virus, and to a lesser extent with corresponding mAb-resistant virus variants. The binding of affinity isolated bovine and swine antibodies to virus was specifically inhibited by the homologous a-IdAb, and in addition, both were capable of neutralizing FMDV in suckling mouse protection and plaque reduction neutralization assays. Therefore, by means of a-IdAb probes generated against FMDV murine Id, two neutralizing Id were identified in bovine and swine. These results suggest that FMDV-neutralizing epitopes recognized by murine systems play a role in the overall immunity of foot-and-mouth disease-susceptible animals.     

Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication

The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.     

Evaluation of the use of transplanted Nassarius reticulatus (Linnaeus, 1758), in monitoring TBT pollution,within the European Water Framework Directive

The European Water Framework Directive (WFD) requires monitoring programmes, to establish a coherent and comprehensive overview of ecological and chemical water status. Recently and within this context, the use of biomarkers has been proposed for incorporation into WFD monitoring programmes. In the present study, cages have been used to transplant (ranging between 14 and 75 days) adults of the dogwhelk Nassarius reticulatus in relation to a gradient of tributyltin (TBT) pollution; this is in order to evaluate its usefulness within the WFD monitoring programmes. Only the most polluted site (close to a shipyard) shows evidence of TBT contamination effects. As such, it may be concluded that the use of caged transplants with N. reticulatus (adults) is useful only for areas of high TBT pollution. Thus, within the European WFD context, this approach is generally not useful for surveillance and operational monitoring, but has some potential in investigative monitoring.     

The importance of the intensity and frequency of perturbations on the germination delay

An iterative model is developed to evaluate the reproductive strategies of plants in environments with different frequencies and intensities of disturbance. Two extreme reproductive strategies are compared: the "homocarpic" strategy, in which all the seeds germinate the following spring, without dormancy, and the "heterocarpic" strategy, whereby, each year, half of the existing seeds germinate. It is observed that this sort of heterocarpy is beneficial in environments with strong perturbations, in which a high percentage of the population dies. However, the frequency of such perturbations does not cause changes in the advantage of one strategy over the other. The results suggest that the intensity of the disturbance is much more important than the frequency.     

New insights into the circadian rhythm of acute myocardial infarction in subgroups

The aim of this study was to determine the existence of the circadian rhythm (CR) in the onset of acute myocardial infarction (AMI) in different patient subgroups. Information was collected about 41,244 infarctions from the database of the ARIAM (Analysis of Delay in AMI) Spanish multicenter study. CR in AMI were explored in subgroups of cases categorized by age, gender, previous ischemic heart disease (PIHD), outcome in coronary care unit, infarction electrocardiograph (ECG) characteristics (Q wave or non-Q wave), and location of AMI. Cases were classified according to these variables in the different subgroups. To verify the presence of CR, a simple test of equality of time series based on the multiple-sinusoid (24, 12, and 8 h periods) cosinor analysis was developed. For the groups as a whole, the time of pain onset as an indicator of the AMI occurrence showed a CR (p<0.0001), with a morning peak at 10:10 h. All the analyzed subgroups also showed CR. Comparison between subgroups showed significant differences in the PIHD (p<0.01) and infarction ECG characteristics (p<0.01) groups. The CR of the subgroup with Q-wave infarction differed from that of non-Q wave subgroup (p<0.01) when the patients had PIHD (23% in Q wave infarction vs. 39.2% in non-Q wave). AMI onset followed a CR pattern, which is also observed in all analyzed subgroups. Differences in the CR according to the Q/non-Q wave infarction characteristics could be determined by PIHD. The cosinor model fit with three components (24, 12, and 8 h periods) showed a higher sensitivity than the single 24 h period analysis.