Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments

Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a 'coiled-coil'-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent K(D) of approx. 0.5 muM and a stoichiometry of approx. 5:1 [actin/C(1-498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.     

Transplantation of mesenchymal stromal cells on mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation

BACKGROUND: Tissue engineering using mesenchymal stromal cells (MSC) represents a promising approach for bone regeneration. Nevertheless, the optimal constructs have yet to be determined. It still remains unclear if there is a benefit of in vitro differentiation of MSC prior to transplantation or if undifferentiated MSC hold the optimal potential concerning new tissue formation. METHODS: After isolation and in vitro expansion, MSC were seeded on mineralized collagen sponges and transplanted in a heterotopic SCID mice model (n=12). While group A contained undifferentiated MSC, in group B cells were cultivated for 14 days in vitro under osteogenic conditions prior to implantation. Results were compared with non-loaded scaffolds (group C). Animals were killed for investigation at 4 and at 8 weeks. RESULTS: In situ hybridization demonstrated integration of MSC for up to 8 weeks in groups A and B. Histology revealed significantly more extracellular matrix synthesis in MSC-seeded scaffolds containing calcium phosphate and collagen type I at 4 and 8 weeks after transplantation compared with unloaded controls. At a biochemical level, higher levels of specific alkaline phosphatase expression were detected in MSC-loaded scaffolds (P<0.05). Scaffolds containing undifferentiated and differentiated MSC did not appear to differ in terms of matrix synthesis and protein expression, while the number of avital cells was significant higher in those probes loaded with differentiated MSC (P<0.01). DISCUSSION: The integration of transplanted cells and MSC-associated matrix synthesis encourages the use of MSC-loaded mineralized collagen for tissue engineering of bone. Furthermore, our data suggest that in vitro differentiation of MSC does not have a positive influence in terms of improved matrix synthesis.     

Toll-like Receptor 4 Is Expressed on Intestinal Stem Cells and Regulates Their Proliferation and Apoptosis via the p53 Up-regulated Modulator of Apoptosis

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.     

Decreases in plasma membrane Ca2+‐ATPase in brain synaptic membrane rafts from aged rats

Precise regulation of free intracellular Ca²⁺ concentrations [Ca²⁺]_i is critical for normal neuronal function, and alterations in Ca²⁺ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca²⁺]_i is the plasma membrane Ca²⁺‐ATPase (PMCA), the high‐affinity transporter that fine tunes the cytosolic nanomolar levels of Ca²⁺. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non‐raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age‐related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.     

Conantokins derived from the Asprella clade impart conRl-B, an N-methyl d-aspartate receptor antagonist with a unique selectivity profile for NR2B subunits

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.     

Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.     

Prothrombotic effects of tumor necrosis factor alpha in vivo are amplified by the absence of TNF-alpha receptor subtype 1 and require TNF-alpha receptor subtype 2

Introduction

Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo.

Methods

Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry.

Results

In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects.

Conclusions

TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.     

Feasibility of intensity-modulated and image-guided radiotherapy for functional organ preservation in locally advanced laryngeal cancer

Purpose

The study aims to assess the feasibility of intensity-modulated and image-guided radiotherapy (IMRT, and IGRT, respectively) for functional preservation in locally advanced laryngeal cancer. A retrospective review of 27 patients undergoing concurrent chemoradiation for locally advanced laryngeal cancers (8 IMRT, 19 IGRT) was undertaken. In addition to regular clinical examinations, all patients had PET imaging at 4 months and 10 months after radiotherapy, then yearly. Loco-regional control, speech quality and feeding-tube dependency were assessed during follow-up visits.

Results

At a median follow-up of 20 months (range 6–57 months), four out of 27 patients (14.8%) developed local recurrence and underwent salvage total laryngectomy. One patient developed distant metastases following salvage surgery. Among the 23 patients who conserved their larynx with no sign of recurrence at last follow-up, 22 (95%) reported normal or near normal voice quality, allowing them to communicate adequately. Four patients (14.8%) had long-term tube feeding-dependency because of severe dysphagia (2 patients) and chronic aspiration (2 patients, with ensuing death from aspiration pneumonia in one patient).

Conclusions and Clinical Relevance

Functional laryngeal preservation is feasible with IMRT and IGRT for locally advanced laryngeal cancer. However, dysphagia and aspiration remain serious complications, due most likely to high radiation dose delivery to the pharyngeal musculatures.     

Using grizzly bears to assess harvest-ecosystem tradeoffs in salmon fisheries

Implementation of ecosystem-based fisheries management (EBFM) requires a clear conceptual and quantitative framework for assessing how different harvest options can modify benefits to ecosystem and human beneficiaries. We address this social-ecological need for Pacific salmon fisheries, which are economically valuable but intercept much of the annual pulse of nutrient subsidies that salmon provide to terrestrial and aquatic food webs. We used grizzly bears, vectors of salmon nutrients and animals with densities strongly coupled to salmon abundance, as surrogates for "salmon ecosystem" function. Combining salmon biomass and stock-recruitment data with stable isotope analysis, we assess potential tradeoffs between fishery yields and bear population densities for six sockeye salmon stocks in Bristol Bay, Alaska, and British Columbia (BC), Canada. For the coastal stocks, we find that both bear densities and fishery yields would increase substantially if ecosystem allocations of salmon increase from currently applied lower to upper goals and beyond. This aligning of benefits comes at a potential cost, however, with the possibility of forgoing harvests in low productivity years. In contrast, we detect acute tradeoffs between bear densities and fishery yields in interior stocks within the Fraser River, BC, where biomass from other salmon species is low. There, increasing salmon allocations to ecosystems would benefit threatened bear populations at the cost of reduced long-term yields. To resolve this conflict, we propose an EBFM goal that values fisheries and bears (and by extension, the ecosystem) equally. At such targets, ecosystem benefits are unexpectedly large compared with losses in fishery yields. To explore other management options, we generate tradeoff curves that provide stock-specific accounting of the expected loss to fishers and gain to bears as more salmon escape the fishery. Our approach, modified to suit multiple scenarios, provides a generalizable method to resolve conflicts over shared resources in other systems.