Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity

14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells, which play an important role in a multitude of signaling pathways. 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner. More than 200 14-3-3 binding partners have been found that are involved in cell cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. A phosphorylation-independent interaction has been reported to occur between 14-3-3 and a C-terminal domain within exoenzyme S (ExoS), a bacterial ADP-ribosyltransferase toxin from Pseudomonas aeruginosa. In this study, we have investigated the effect of amino acid mutations in this C-terminal domain of ExoS on ADP-ribosyltransferase activity and the 14-3-3 interaction. Our results suggest that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity, as cytotoxicity analysis reveals that substitution of this leucine significantly weakens the ability of ExoS to mediate cell death. Leucine-428 is also required for the ability of ExoS to modify the eukaryotic endogenous target Ras. Finally, single amino acid substitutions of positions 426-428 reduce the interaction potential of 14-3-3 with ExoS in vitro.     

Microbial Fossils in the 2.63 Ga Jeerinah Formation,Western Australia—Evidence of Microbial Oxidation

A diamond drill core from the upper part of the Jeerinah Formation (～2.63 Ga), underlying the Hamersley Group, deposited at a time when the oxygen concentrations in the marine environment were extremely low, was examined for microbial fossils. The paper presents organo-mineral structures in the form of twisted stalks produced by bacteria being present in the laminated black carbonaceous shale sediments. These twisted stalks are organo-mineral structures produced by microaerophilic Fe(II)-oxidizing-type bacteria such as Gallionella and/or Mariprofundus that are active at very low-oxygen concentrations, thus providing evidence for oxygen being present in the marine environment at 2.63 Ga.     

The neuroblastoma ALK(I1250T) mutation is a kinase-dead RTK in vitro and in vivo

Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations-ALK(I1250T)-identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALK(I1250T) mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALK(I1250T) and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALK(I1250T) is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALK(F1174S) mutant. Identical results were obtained when the ALK(I1250T) mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALK(I1250T) mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.     

Differences in early and late responses between neurotrophin-stimulated trkA- and trkC-transfected SH-SY5Y neuroblastoma cells.

Despite their sympathetic neuroblast origin, highly malignant neuroblastoma tumors and derived cell lines have no or low expression of the neurotrophin receptor genes, trkA and trkC. Expression of exogenous trkA in neuroblastoma cells restores their ability to differentiate in response to nerve growth factor (NGF). Here we show that stable expression of trkC in SH-SY5Y neuroblastoma cells resulted in morphological and biochemical differentiation upon treatment with neurotrophin-3 (NT-3). To some extent, trkA- and trkC-transfected SH-SY5Y (SH-SY5Y/trkA and SH-SY5Y/trkC) cells resembled one another in terms of early signaling events and neuronal marker gene expression, but important differences were observed. Although induced Erk 1/2 and Akt/PKB phosphorylation was stronger in NT-3-stimulated SH-Y5Y/trkC cells, activation of the immediate-early genes tested was more prominent in NGF-treated SH-SY5Y/ trkA cells. In particular, c-fos was not induced in the SH-SY5Y/trkC cells. There were also phenotypic differences. The concentrations of norepinephrine, the major sympathetic neurotransmitter, and growth cone-located synaptophysin, a neurosecretory granule protein, were increased in NGF-treated SH-SY5Y/trkA but not in NT-3-treated SH-SY5Y/trkC cells. Our data suggest that NT-3/p145trkC and NGF/p140trkA signaling differ in some aspects in neuroblasoma cells, and that this may explain the phenotypic differences seen in the long-term neurotrophin-treated cells.     

Feeding ecology of juvenile turbot Scophthalmus maximus and flounder Pleuronectes flesus at Gotland, Central Baltic Sea

Food and feeding of juvenile turbot Scophthalmus maximus and flounder Pleuronectes flesus were studied in five nursery areas at Gotland, Central Baltic Sea, ICES SD 27 and SD 28. Ontogeny involved partitioning of available food resources. The food choice of turbot <30 mm standard length (L_S) included both planktonic‐hyperbenthic prey (calanoid copepods and mysids) and epibenthic–endobenthic prey (chironomids and amphipods), whereas turbot ≥30 mm L_S fed mainly on hyperbenthic species (mysids and fishes). Conversely, for flounder, epibenthic–endobenthic prey were the most abundant prey items throughout development (harpactocoid copepods, oligochaetes and chironomids for fish <40 mm L_S and oligochaetes, chironomids and amphipods for flounder ≥40 mm L_S). Thus, the highest degree of dietary overlap occurred between turbot <30 mm and flounder ≥40 mm. Food composition for both turbot and flounder varied, however, according to exposure and predominant wind direction in the nursery area. For example, expressed as the ratio between the biomass of mysids and fishes consumed, the relative importance of mysids v. fishes as food source for turbot, varied from <1 in the most sheltered area to 16 and 27 in the more open areas. Considerable differences in feeding incidence were recorded; mean ±s .d . 58 ± 20% for turbot <30 mm L_S and 83 ± 8% for turbot ≥30 mm L_S, as opposed to ≥85–90% for flounder irrespective of size. The lower feeding success of turbot <30 mm L_S was related to mysid abundance, shown to vary spatially and temporally, and to density of flounder, indicating that food availability, and potentially interspecific competition, influence feeding of early juvenile turbot with implications for survival following settlement. Regarding variability in abundance, hyperbenthic prey, as mysids, are considered more variable than epi‐ and endobenthic organisms. Hence, in addition to the ‘nursery size hypothesis’, i.e. the positive relationship between abundance of recruits and extension of nursery areas, variability in food availability may explain the average lower recruitment of turbot as compared to other flatfishes, e.g. flounder.     

Compound Eyes of Some Deep-Sea and Fiord Mysid Crustaceans

The compound eyes of the deep-sea mysid Boreomysis scyphops and the two mysid species Amblyops abbreviata and Pseudomma affine, which are indigenous to deep fiords in Norway, have been investigated. The eye stalks are greatly transformed, but contain hypertrophied retinas. The ommatidia of all three species lack a dioptric apparatus, possessing only retinular cells, which are arranged in a cylinder-like fashion. Folds from the retinular cells project into the “cylinder” and are covered with microvilli. The arrangement is explained functionally by an increase in the photopigment-bearing surface as an adaptation to low-light intensities. The orderly arrangement of microvilli in most arthropod compound eyes has been lost, and the arrangement is thus multidirectional in these mysids. With the photopigment dipoles arranged along the microvillar axis, the disorderly arrangement of microvilli signifies a more efficient capture of non-polarized light. It is concluded that the mysid compound eyes show adaptations to low-light intensities probably acquired during the species' evolutionary descent into deep-sea habitats. Amblyops abbreviata and Pseudomma affine, belonging to genera with entirely transformed eyes and with an ultrastructure less “normal” than that of Boreomysis scyphops are believed to be earlier descendants into the deep-sea habitats than the latter species, which belongs to a genus in which most of the species have well-developed eyes.     

Microbial communities in a porphyry copper tailings impoundment and their impact on the geochemical dynamics of the mine waste

The distribution and diversity of acidophilic bacteria of a tailings impoundment at the La Andina copper mine, Chile, was examined. The tailings have low sulfide (1.7% pyrite equivalent) and carbonate (1.4% calcite equivalent) contents and are stratified into three distinct zones: a surface (0-70-80 cm) 'oxidation zone' characterized by low-pH (2.5-4), a 'neutralization zone' (70-80 to 300-400 cm) and an unaltered 'primary zone' below 400 cm. A combined cultivation-dependent and biomolecular approach (terminal restriction enzyme fragment length polymorphism and 16S rRNA clone library analysis) was used to characterize the indigenous prokaryotic communities in the mine tailings. Total cell counts showed that the microbial biomass was greatest in the top 125 cm of the tailings. The largest numbers of bacteria (10(9) g(-1) dry weight of tailings) were found at the oxidation front (the junction between the oxidation and neutralization zones), where sulfide minerals and oxygen were both present. The dominant iron-/sulfur-oxidizing bacteria identified at the oxidation front included bacteria of the genus Leptospirillum (detected by molecular methods), and Gram-positive iron-oxidizing acidophiles related to Sulfobacillus (identified both by molecular and cultivation methods). Acidithiobacillus ferrooxidans was also detected, albeit in relatively small numbers. Heterotrophic acidophiles related to Acidobacterium capsulatum were found by molecular methods, while another Acidobacterium-like bacterium and an Acidiphilium sp. were isolated from oxidation zone samples. A conceptual model was developed, based on microbiological and geochemical data derived from the tailings, to account for the biogeochemical evolution of the Piuquenes tailings impoundment.     

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G)

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.     

Exoenzyme S of Pseudomonas aeruginosa is not able to induce apoptosis when cells express activated proteins, such as Ras or protein kinase B/Akt

Intracellular targeting of the Pseudomonas aeruginosa toxins, such as exoenzyme S (ExoS), cause cell death, as well as morphological and physiological changes in various tissue culture cells and animal models. In this report we have investigated the mechanism behind ExoS-mediated cell death. In order to address this issue, we have used cell lines expressing activated forms of various components of the Ras signalling pathway in order to evaluate the importance of the Ras pathway for viability and survival upon ExoS infection. Here we show that activated Ras is able to protect cells against cell death, regardless of whether it has been ADP-ribosylated by ExoS. Further, an activated form of protein kinase B (PKB)/Akt also leads to decreased level of cell death in response to ExoS infection, indicating that an important ExoS survival target is located upstream of Raf-1 and PKB/Akt. Moreover, we show that ExoS infection inhibits phosphorylation of FOXO3a, and induces caspase-3 activity, which are hallmarks for induction of cell death. In conclusion, we suggest that Ras proteins are an important cellular target for the P. aeruginosa toxin ExoS, which induces cell death during pathogenesis as a means of defending the bacterium against eukaryotic phagocytosis.