Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders.

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interac-tion between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M.pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All pecombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclon-al antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M.pneumoniae infections by interaction with human plasminogen.

The role of competition in tree communities is increasingly well understood, while little is known about the patterns and mechanisms of the interplay between above- and belowground competition in tree communities. This knowledge, however, is crucial for a better understanding of community dynamics and developing adaptive near-natural management strategies. We assessed neighbourhood interactions in an unmanaged old-growth European beech (Fagus sylvatica) forest by quantifying variation in the intensity of above- (shading) and belowground competition (crowding) among dominant and co-dominant canopy beech trees during tree maturation. Shading had on average a much larger impact on radial growth than crowding and the sensitivity to changes in competitive conditions was lowest for crowding effects. We found that each mode of competition reduced the effect of the other. Increasing crowding reduced the negative effect of shading, and at high levels of shading, crowding actually had a facilitative effect and increased growth. Our study demonstrates that complementarity in above- and belowground processes enable F. sylvatica to alter resource acquisition strategies, thus optimising tree radial growth. As a result, competition seemed to become less important in stands with a high growing stock and tree communities with a long continuity of anthropogenic undisturbed population dynamics. We suggest that growth rates do not exclusively depend on the density of potential competitors at the intraspecific level, but on the conspecific aggregation of large-diameter trees and their functional role for regulating biotic filtering processes. This finding highlights the potential importance of the rarely examined relationship between the spatial aggregation pattern of large-diameter trees and the outcome of neighbourhood interactions, which may be central to community dynamics and the related forest ecosystem services.

Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome.

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21 - pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program.

“Neglected Rickettsiaceae” (i.e. those harboured by non-hematophagous eukaryotic hosts) display greater phylogenetic variability and more widespread dispersal than pathogenic ones; yet, the knowledge about their actual host range and host shift mechanism is scarce. The present work reports the characterization following the full-cycle rRNA approach (SSU rRNA sequence, specific in situ hybridization, and ultrastructure) of a novel rickettsial bacterium, herewith proposed as 'Candidatus Megaira polyxenophila' gen. nov., sp. nov. We found it in association with four different free-living ciliates (Diophrys oligothrix, Euplotes octocarinatus, Paramecium caudatum, and Spirostomum sp., all belonging to Alveolata, Ciliophora); furthermore it was recently observed as intracellular occurring in Carteria cerasiformis and Pleodorina japonica (Chlorophyceae, Chlorophyta). Phylogenetic analyses demonstrated the belonging of the candidate new genus to the family Rickettsiaceae (Alphaproteobacteria, Rickettsiales) as a sister group of the genus Rickettsia. In situ observations revealed the ability of the candidate new species to colonize either nuclear or cytoplasmic compartments, depending on the host organism. The presence of the same bacterial species within different, evolutionary distant, hosts indicates that 'Candidatus Megaira polyxenophila' recently underwent several distinct host shifts, thus suggesting the existence of horizontal transmission pathways. We consider these findings as indicative of an unexpected spread of rickettsial infections in aquatic communities, possibly by means of trophic interactions, and hence propose a new interpretation of the origin and phylogenetic diversification of rickettsial bacteria.

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.