Two functionally distinct Axin-like proteins regulate canonical Wnt signaling in C. elegans

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.     

The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity

The lack of trehalose accumulation in most plant species has been partly attributed to the presence of an active trehalase. Although trehalose synthesis enzymes are thought to be cytosolic, and previous studies have indicated that trehalase activity is extracellular, the exact location of the enzyme has not yet been established in plant cell. We present evidence that the yet uncharacterised full-length Arabidopsis trehalase is a plasma membrane-bound protein, probably anchored to the membrane through a predicted N-terminal membrane spanning domain. The full-length AtTRE1, when expressed in yeast can functionally substitute for the extracellularly active trehalase Ath1p, by sustaining the growth of an ath1 null mutant strain on trehalose and at pH 4.8. We further demonstrate that AtTRE1 expressed in yeast is plasma membrane-bound as in plant cell. In light of these findings, the regulation of plant cell endogenous trehalose by trehalase is discussed.     

Tranexamic acid suppresses the release of mitochondrial DNA,protects the endothelial monolayer and enhances oxidative phosphorylation

Damage-associated molecular patterns, including mitochondrial DNA (mtDNA) are released during hemorrhage resulting in the development of endotheliopathy. Tranexamic acid (TXA), an antifibrinolytic drug used in hemorrhaging patients, enhances their survival despite the lack of a comprehensive understanding of its cellular mechanisms of action. The present study is aimed to elucidate these mechanisms, with a focus on mitochondria. We found that TXA inhibits the release of endogenous mtDNA from granulocytes and endothelial cells. Furthermore, TXA attenuates the loss of the endothelial monolayer integrity induced by exogenous mtDNA. Using the Seahorse XF technology, it was demonstrated that TXA strongly stimulates mitochondrial respiration. Studies using Mitotracker dye, cells derived from mito-QC mice, and the ActivSignal IPAD assay, indicate that TXA stimulates biogenesis of mitochondria and inhibits mitophagy. These findings open the potential for improvement of the strategies of TXA applications in trauma patients and the development of more efficient TXA derivatives.     

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain–containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain–containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD–Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3–STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.     

Closed state of both binding domains of homodimeric mGlu receptors is required for full activity

Membrane receptors, key components in signal transduction, often function as dimers. These include some G protein-coupled receptors such as metabotropic glutamate (mGlu) receptors that have large extracellular domains (ECDs) where agonists bind. How agonist binding in dimeric ECDs activates the effector domains remains largely unknown. The structure of the dimeric ECDs of mGlu(1) solved in the presence of agonist revealed two specific conformations in which either one or both protomers are in an agonist-stabilized closed form. Here we examined whether both conformations correspond to an active form of the full-length receptor. Using a system that allows the formation of dimers made of a wild-type and a mutant subunit, we show that the closure of one ECD per dimer is sufficient to activate the receptor, but the closure of both ECDs is required for full activity.     

Relationships among murine CD11c(high) dendritic cell subsets as revealed by baseline gene expression patterns

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.     

Organization of the cytokeratin network in an epithelial cell

The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters.     

Neurovascular congruence results from a shared patterning mechanism that utilizes Semaphorin3A and Neuropilin-1

Peripheral nerves and blood vessels have similar patterns in quail forelimb development. Usually, nerves extend adjacent to existing blood vessels, but in a few cases, vessels follow nerves. Nerves have been proposed to follow vascular smooth muscle, endothelium, or their basal laminae. Focusing on the major axial blood vessels and nerves, we found that when nerves grow into forelimbs at E3.5-E5, vascular smooth muscle was not detectable by smooth muscle actin immunoreactivity. Additionally, transmission electron microscopy at E5.5 confirmed that early blood vessels lacked smooth muscle and showed that the endothelial cell layer lacks a basal lamina, and we did not observe physical contact between peripheral nerves and these endothelial cells. To test more generally whether lack of nerves affected blood vessel patterns, forelimb-level neural tube ablations were performed at E2 to produce aneural limbs; these had completely normal vascular patterns up to at least E10. To test more generally whether vascular perturbation affected nerve patterns, VEGF(165), VEGF(121), Ang-1, and soluble Flt-1/Fc proteins singly and in combination were focally introduced via beads implanted into E4.5 forelimbs. These produced significant alterations to the vascular patterns, which included the formation of neo-vessels and the creation of ectopic avascular spaces at E6, but in both under- and overvascularized forelimbs, the peripheral nerve pattern was normal. The spatial distribution of semaphorin3A protein immunoreactivity was consistent with a negative regulation of neural and/or vascular patterning. Semaphorin3A bead implantations into E4.5 forelimbs caused failure of nerves and blood vessels to form and to deviate away from the bead. Conversely, semaphorin3A antibody bead implantation was associated with a local increase in capillary formation. Furthermore, neural tube electroporation at E2 with a construct for the soluble form of neuropilin-1 caused vascular malformations and hemorrhage as well as altered nerve trajectories and peripheral nerve defasciculation at E5-E6. These results suggest that neurovascular congruency does not arise from interdependence between peripheral nerves and blood vessels, but supports the hypothesis that it arises by a shared patterning mechanism that utilizes semaphorin3A.