Dissection of Host Cell Signal Transduction during Acinetobacter baumannii – Triggered Inflammatory Response

Infected airway epithelial cells up-regulate the expression of chemokines, chiefly IL-8, and antimicrobial molecules including β-defensins (BD). Acinetobacter baumannii is a cause of hospital-acquired pneumonia. We examined whether A. baumannii induced the expressions of IL-8 and BD2 by airway epithelial cells and the receptors implicated in bacterial detection. A549 and human primary airway cells released IL-8 upon infection. A. baumannii-infected cells also increased the expression of BD2 which killed A. baummannii strains. IL-8 induction was via NF-κB and mitogen-activated kinases p38 and p44/42-dependent pathways. A. baumannii engaged Toll-like receptor (TLR) 2 and TLR4 pathways and A549 cells could use soluble CD14 as TLRs co-receptor. A. baumannii lipopolysaccharide stimulated IL-8 release by A549 cells and sCD14 facilitated the recognition of the lipopolysaccharide. Mass spectrometry analysis revealed that A. baumannii lipid A structure matches those with endotoxic potential. These results demonstrate that airway epithelial cells produce mediators important for A. baumannii clearance.     

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.     

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.     

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.     

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.     

Forest Management Considerations for Conservation of Black Woodpecker Dryocopus martius and White-backed Woodpecker Dendrocopos leucotos Populations in Quinto Real (Spanish Western Pyrenees)

The woodlands of Quinto Real (Quinto Real, Erreguerena and Legua Acotada) are a 3,000 hectare beech (Fagus sylvatica) forest managed by the shelterwood system applied to even-aged (regular) stands. This study analyses how forest management determines the local distribution of the white-backed woodpecker (Dendrocopos leucotos) and black woodpecker (Dryocopus martius) and its relationship with the type, structure and size of the stands used for nesting by both species, as well as their dead wood requirements. The most suitable nesting habitat of both species is the mature forest (stands of regular large final crop trees), but the size of the mature fragments and a minimum quantity of dead wood is also important.     

Role of Bacterial Surface Structures on the Interaction of Klebsiella pneumoniae with Phagocytes

Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis.     

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.