Cell Type Specific Signalling by Hematopoietic Growth Factors in Neural Cells

Correct timing and spatial location of growth factor expression is critical for undisturbed brain development and functioning. In terminally differentiated cells distinct biological responses to growth factors may depend on cell type specific activation of signalling cascades. We show that the hematopoietic growth factors thrombopoietin (TPO) and granulocyte colony-stimulating factor (GCSF) exert cell type specific effects on survival, proliferation and the degree of phosphorylation of Akt1, ERK1/2 and STAT3 in rat hippocampal neurons and cortical astrocytes. In neurons, TPO induced cell death and selectively activated ERK1/2. GCSF protected neurons from TPO- and hypoxia-induced cell death via selective activation of Akt1. In astrocytes, neither TPO nor GCSF had any effect on cell viability but inhibited proliferation. This effect was accompanied by activation of ERK1/2 and inhibition of STAT3 activity. A balance between growth factors, their receptors and signalling proteins may play an important role in regulation of neural cell survival.     

Regional myocardial work by strain Doppler echocardiography and LV pressure: a new method for quantifying myocardial function

There is a need for better methods to quantify regional myocardial function. In the present study, we investigated the feasibility of quantifying regional function in terms of a segmental myocardial work index as derived from strain Doppler echocardiography (SDE) and invasive pressure. In 10 anesthetized dogs, we measured left ventricular (LV) pressure by micromanometer and myocardial longitudinal strains by SDE and sonomicrometry. The regional myocardial work index (RMWI) was calculated as the area of the pressure-strain loop. As a reference method for strain, we used sonomicrometry. By convention, the loop area was assigned a positive sign when the pressure-strain coordinates rotated counterclockwise. Measurements were done at baseline and during volume loading and left anterior descending coronary artery (LAD) occlusion, respectively. There was a good correlation between RMWI calculated from strain by SDE and strain by sonomicrometry (y = 0.73x + 0.21, r = 0.82, P < 0.01). Volume loading caused an increase in RMWI from 1.3 +/- 0.2 to 2.2 +/- 0.1 kJ/m3 (P < 0.05) by SDE and from 1.5 +/- 0.3 to 2.7 +/- 0.3 kJ/m3 (P = 0.066) by sonomicrometry. Short-term ischemia (1 min) caused a decrease in RMWI from 1.3 +/- 0.2 to 0.3 +/- 0.04 kJ/m3 (P < 0.05) and from 1.3 +/- 0.3 to 0.5 +/- 0.2 kJ/m3 (P < 0.05) by SDE and sonomicrometry, respectively. In the nonischemic ventricle and during short-term ischemia, the pressure-strain loops rotated counterclockwise, consistent with actively contracting segments. Long-term ischemia (3 h), however, caused the pressure-strain loop to rotate clockwise, consistent with entirely passive segments, and the loop areas became negative, -0.2 +/- 0.1 and -0.1 +/- 0.03 kJ/m3 (P < 0.05) by SDE and sonomicrometry, respectively. A RMWI can be estimated by SDE in combination with LV pressure. Furthermore, the orientation of the loop can be used to assess whether the segment is active or passive.     

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Increasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of E. faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especially l-glutamine and l-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets.     

Critical Evaluation of Phosphate Solubilizing Pseudomonads Isolated from a Partially Recultivated Potash Tailings Pile

Ten phosphate solubilizing pseudomonads isolated from a partially recultivated potash tailings pile in Germany were characterized and tested for their potential to assist in the ongoing recultivation process. Despite fertilization, the plants which are grown for recultivation show phosphate deficiency symptoms, and therefore the isolates are intended to be used as biofertilizer inoculants. On agar plates incubated at five different temperatures, some of the strains showed a temperature-dependent ability to solubilize tricalcium phosphate, while others performed the same at any given temperature. In liquid medium, the isolates solubilized between 271 and 730 μg ml(-1) of phosphate from tricalcium phosphate. Both the weakest (designated S10) and the strongest solubilizing strain (S06) were further tested for their viability during solubilization. In an assay over the course of 1 week, both strains released their maximum amount of phosphate after 2-4 days. At that later point of time, however, viable cells of isolate S06 were no longer detectable, whereas the weaker strain S10 could be cultured after 1 week in broth. Taking all in vitro observations into account, the usability of the isolates as biofertilizers is critically discussed regarding both the in situ conditions on the tailings pile and the lowered viability due to the excess production of organic acids.     

Diversity of Xenorhabdus and Photorhabdus spp. and Their Symbiotic Entomopathogenic Nematodes from Thailand

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand.     

Predator Assemblage Structure and Temporal Variability of Species Richness and Abundance in Forests of High Tree Diversity

Predators significantly affect ecosystem functions, but our understanding of to what extent findings can be transferred from experiments and low‐diversity systems to highly diverse, natural ecosystems is limited. With a particular threat of biodiversity loss at higher trophic levels, however, knowledge of spatial and temporal patterns in predator assemblages and their interrelations with lower trophic levels is essential for assessing effects of trophic interactions and advancing biodiversity conservation in these ecosystems. We analyzed spatial and temporal variability of spider assemblages in tree species‐rich subtropical forests in China, across 27 study plots varying in woody plant diversity and stand age. Despite effects of woody plant richness on spider assemblage structure, neither habitat specificity nor temporal variability of spider richness and abundance were influenced. Rather, variability increased with forest age, probably related to successional changes in spider assemblages. Our results indicate that woody plant richness and theory predicting increasing predator diversity with increasing plant diversity do not necessarily play a major role for spatial and temporal dynamics of predator assemblages in such plant species‐rich forests. Diversity effects on biotic or abiotic habitat conditions might be less pronounced across our gradient from medium to high plant diversity than in previously studied less diverse systems, and bottom‐up effects might level out at high plant diversity. Instead, our study highlights the importance of overall (diversity‐independent) environmental heterogeneity in shaping spider assemblages and, as indicated by a high species turnover between plots, as a crucial factor for biodiversity conservation at a regional scale in these subtropical forests.     

Chromosomal Dynamism in Progeny of Outbreak-Related Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx₂) in the outbreak strain or with the loss of this gene. The two stx₂ alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx₂ genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

A Crystallin Fold in the Interleukin-4-inducing Principle of Schistosoma mansoni Eggs (IPSE/α-1) Mediates IgE Binding for Antigen-independent Basophil Activation

The IL-4-inducing principle from Schistosoma mansoni eggs (IPSE/α-1), the major secretory product of eggs from the parasitic worm S. mansoni, efficiently triggers basophils to release the immunomodulatory key cytokine interleukin-4. Activation by IPSE/α-1 requires the presence of IgE on the basophils, but the detailed molecular mechanism underlying activation is unknown. NMR and crystallographic analysis of IPSEΔNLS, a monomeric IPSE/α-1 mutant, revealed that IPSE/α-1 is a new member of the βγ-crystallin superfamily. We demonstrate that this molecule is a general immunoglobulin-binding factor with highest affinity for IgE. NMR binding studies of IPSEΔNLS with the 180-kDa molecule IgE identified a large positively charged binding surface that includes a flexible loop, which is unique to the IPSE/α-1 crystallin fold. Mutational analysis of amino acids in the binding interface showed that residues contributing to IgE binding are important for IgE-dependent activation of basophils. As IPSE/α-1 is unable to cross-link IgE, we propose that this molecule, by taking advantage of its unique IgE-binding crystallin fold, activates basophils by a novel, cross-linking-independent mechanism.     

Transduction of a Plasmid Carrying the Cohesive End Region from Lactococcus lactis Bacteriophage ΦLC3

Plasmids carrying the cohesive end region from temperate lactococcal bacteriophage ΦLC3 could be packaged in vivo by ΦLC3 and transduced into its host strain, Lactococcus lactis subsp. cremoris NCDO 1201. The transduction frequencies were between 10^-4 and 10^-3 transducing particles per PFU, depending on the size of the phage DNA insert. This transduction system is limited to only certain lactococcal strains. The ΦLC3 cohesive site region (cos) appears to play an important role in plasmid transduction.