Identification and characterization of cells with high angiogenic potential and transitional phenotype in calcific aortic valve

Recent data suggest that angiogenesis plays an important role in the pathogenesis of valvular disease. However, the cellular mechanisms underlying this process remain unknown. This study aimed at identifying and characterizing the cellular components responsible for pathological neovascularization in calcific aortic valves (CAV). Immunohistochemical analysis of uncultured CAV tissues revealed that smooth muscle alpha-actin (alpha-SMA)-positive cells, which coexpressed Tie-2 and vascular endothelial growth factor receptor-2 (VEGFR-2), can be identified prior to the initiation of capillary-like tube formation. In a second step, leaflets of CAV and non-calcific aortic valves (NCAV) were cultured and the cells involved in capillary-like tube formation were isolated. The majority of these cells displayed the same phenotype as non-cultured cells identified in CAV tissues, i.e., expression of alpha-SMA, Tie-2, and VEGFR-2. In comparison to cells isolated from cultures of NCAV leaflets, these cells showed enhanced angiogenic activity as demonstrated by migration and tube assays. The coexpression of VEGFR-2 and Tie-2 together with alpha-SMA suggests both endothelial and mesenchymal properties of the angiogenically activated cells involved in valvular neovascularization. Hence, our findings might provide new insights into the process of pathological angiogenesis in cardiac valves.     

The WD repeat protein FAN regulates lysosome size independent from abnormal downregulation/membrane recruitment of protein kinase C

FAN (factor associated with neutral sphingomyelinase [N-SMase] activation) exhibits striking structural homologies to Lyst (lysosomal trafficking regulator), a BEACH protein whose inactivation causes formation of giant lysosomes/Chediak-Higashi syndrome. Here, we show that cells lacking FAN show a statistically significant increase in lysosome size (although less pronounced as Lyst), pointing to previously unrecognized functions of FAN in regulation of the lysosomal compartment. Since FAN regulates activation of N-SMase in complex with receptor for activated C-kinase (RACK)1, a scaffolding protein that recruits and stabilizes activated protein kinase C (PKC) isotypes at cellular membranes, and since an abnormal (calpain-mediated) downregulation/membrane recruitment of PKC has been linked to the defects observed in Lyst-deficient cells, we assessed whether PKC is also of relevance in FAN signaling. Our results demonstrate that activation of PKC is not required for regulation of N-SMase by FAN/RACK1. Conversely, activation of PKC and recruitment/stabilization by RACK1 occurs uniformly in the presence or absence of FAN (and equally, Lyst). Furthermore, regulation of lysosome size by FAN is not coupled to an abnormal downregulation/membrane recruitment of PKC by calpain. Identical results were obtained for Lyst, questioning the previously reported relevance of PKC for formation of giant lysosomes and in Chediak-Higashi syndrome. In summary, FAN mediates activation of N-SMase as well as regulation of lysosome size by signaling pathways that operate independent from activation/membrane recruitment of PKC.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

The genotype specific competitive ability does not correlate with infection in natural Daphnia magna populations

Background

Different evolutionary hypotheses predict a correlation between the fitness of a genotype in the absence of infection and the likelihood to become infected. The cost of resistance hypothesis predicts that resistant genotypes pay a cost of being resistant and are less fit in the absence of parasites. The inbreeding-infection hypothesis predicts that the susceptible individuals are less fit due to inbreeding depression.

Methods and Results

Here we tested if a host''s natural infection status was associated with its fitness. First, we experimentally confirmed that cured but formerly infected Daphnia magna are genetically more susceptible to reinfections with Octosporea bayeri than naturally uninfected D. magna. We then collected from each of 22 populations both uninfected and infected D. magna genotypes. All were treated against parasites and kept in their asexual phase. We estimated their relative fitness in an experiment against a tester genotype and in another experiment in direct competition. Consistently, we found no difference in competitive abilities between uninfected and cured but formerly infected genotypes. This was the case both in the presence as well as in the absence of sympatric parasites during the competition trials.

Conclusions

Our data do not support the inbreeding-infection hypothesis. They also do not support a cost of resistance, however ignoring other parasite strains or parasite species. We suggest as a possible explanation for our results that resistance genes might segregate largely independently of other fitness associated genes in this system.     

Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells

Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrP(Sc)). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrP(C)) has been reported to abolish PrP(Sc) infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrP(C), active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP-virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrP(C) in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrP(Sc) in prion-infected cells. If also effective in vivo, PrP-virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion-mediated diseases.     

Raptors breeding on weaver nests in trees and on man-made structures

Several raptor species nest on top of large weaver nests. These weaver nests are usually sited in trees, but 11.7% of Red-billed Buffalo-Weaver Bubalornis niger and 25.7% of Sociable Weaver Philetairus socius nests occur on man-made structures. In an extensive literature search, a total of 16 raptor species were recorded as nesting on top of weaver nests. At least 10 raptor species used weaver nests built in trees. Seven raptor species used weaver nests on man-made sites and four raptor species only used weaver nests built on man-made sites. No owls have been recorded as using weaver nests on man-made sites. There are historical records of raptors nesting on top of weaver nests in trees, while nesting on top of weaver nests sited on man-made structures appears to be a more recent adaptation. Costs and benefits of nesting on man-made sites are briefly listed. Nesting on man-made sites may increase (by both weavers and raptors) and raptor researchers are encouraged to document cases of raptors nesting on weaver nests where these are placed in trees or artificial sites, so that there is a record of changes in frequency of nest site usage by raptors.     

Disease‐linked TDP‐43 hyperphosphorylation suppresses TDP‐43 condensation and aggregation

Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation.