More insights into the CCN3/Connexin43 interaction complex and its role for signaling

Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C‐terminus. We have previously demonstrated that transfection of Cx43 leads to significantly reduced proliferation of placental tumor cells through upregulating and binding of the growth regulator CCN3 (NOV) at the C‐terminus of Cx43. Here, we combined fluorescence resonance energy transfer (FRET), co‐immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild‐type Cx43 (amino acids 1‐382) and with mutants of Cx43 truncated at the C‐terminus resulting in Cx43 proteins of amino acids 1‐374, 1‐273, 1‐264, 1‐257 in 293T cells. These results matched the co‐immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild‐type Cx43 and one deletion construct (1‐374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C‐terminus of Cx43 interacting with CCN3. J. Cell. Biochem. 110: 129–140, 2010. © 2010 Wiley‐Liss, Inc.     

(4-Piperidinylphenyl)aminoethyl amides as a novel class of non-covalent cathepsin K inhibitors

A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.     

Water flux through human aquaporin 1: inhibition by intracellular furosemide and maximal response with high osmotic gradients

This work studies water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, applying a technique where cellular content is replaced with a known medium, with the possibility of measuring intracellular pressure. Consequences on water transport—produced by well-known anisotonic gradients and by the intracellular effect of probable aquaporin inhibitors—were tested. In this way, the specific intracellular inhibition of hAQP1 by the diuretic drug furosemide was demonstrated. In addition, experiments imposing anisotonic mannitol gradients with a constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response when the gradient is higher than 150 mosmol kg_W⁻¹. These results would indicate that the passage of water molecules through hAQP1 may have a maximum rate. As a whole, this work demonstrates the technical advantage of controlling both intracellular pressure and medium composition in order to study biophysical properties of hAQP1, and contributes information on water channel behavior under osmotic challenges and the discovery of new inhibitors.     

Direct and indirect effects of ski run management on alpine Orthoptera

Alpine landscapes are heavily influenced by ski run management, which can have severe impacts on alpine biodiversity. To assess these impacts on alpine Orthoptera, we compared species richness and species abundance in 41 plot pairs on ski runs and adjacent off-slope control plots in three ski resorts in Austria and Germany. A mixed modelling approach was used to assess the impacts of ski run preparation, artificial snow-making and environmental variables such as altitude, cover of dwarf shrubs and the application of fertilizer. Ski run plots showed a significantly lower species richness and number of individuals than control plots. Moreover, artificial snow led to a further decrease in species number. Hierarchical variance partitioning revealed that Orthoptera community composition is best predicted by environmental variables indirectly related to ski run management (fertilization, cover of dwarf shrubs) and to altitude. Only one out of five species significantly decreased in abundance after artificial snow-making. Other species were more sensitive to fertilizing and altitude. Dwarf shrubs were negatively associated with ski run management but positively associated with abundance of three species and species richness. Our data provide evidence for both direct and indirect consequences of ski runs and artificial snow-making on alpine Orthoptera. Overall, Orthoptera communities are suitable indicators for human-induced changes in alpine environments. In particular, a shift towards generalist species such as Chorthippus parallelus along with a decrease in typical alpine species gives cause for concern as this implies a homogenisation of biodiversity owing to ski run management.     

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.     

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d’ être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection.     

Evaluating mortality rates and causalities in a critically endangered felid across its whole distribution range

The conservation of endangered species requires accurate data, and knowledge of cause-specific mortality rates is one of the most important issues. In recent years, conservation programs for the critically endangered Iberian lynx Lynx pardinus have been developed on the basis of mortality data derived 30 years ago from the small Doñana population. Thus, there is an urgent need for an update of mortality rates and causes in both populations (Sierra Morena and Doñana). Here we use radio-tracking information from the whole range of the Iberian lynx to quantify mortality rates and identify their causes. Between 2006 and 2011, we radio-tagged 78 Iberian lynxes from its two remaining populations (39 from Sierra Morena and 39 from Doñana). Mortality events were evaluated to identify causes, and cause-specific annual mortality rates (AMR) were obtained using the nonparametric cumulative incidence function estimator. Overall, AMR was estimated at 0.16?±?0.05 (0.19?±?0.09 in Sierra Morena and 0.12?±?0.07 in Doñana). Disease was the main cause of mortality both for the whole population and the Doñana population. Poaching was the main cause of mortality in Sierra Morena. Our results suggest that the best strategy for conserving this species is to focus action on decreasing the fatal effect of disease and poaching. Given the possible existence of an underlying inbreeding-mediated immunosuppression, genetic management aimed at increasing the genetic diversity of this population is also recommended.