The discovery of novel calcium sensing receptor negative allosteric modulators

The design and profile of a series of zwitterionic calcium sensing receptor negative allosteric modulators is described. Evaluation of key analogues using a rat model demonstrate a robust response, significantly improved potency over ronacaleret and have the potential as an oral, anabolic treatment for osteoporosis.     

FERM domain phosphoinositide binding targets merlin to the membrane and is essential for its growth-suppressive function

The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP(2), via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin.     

Balancing bioenergy and biosecurity policies: estimating current and future climate suitability patterns for a bioenergy crop

In an apparent paradox, bioenergy crops offer potential benefits to a world adjusting to the challenges of climate change and declining fossil fuel stocks, as well as potential ecological and economic threats resulting from biological invasions. In considering this paradox it is important to understand that benefits and threats may not always be apparent in equal measure throughout the potential range of each candidate biofuel species. In some environments, a species could potentially produce valuable biological materials without posing a significant invasion threat. In this study, we develop a bioclimatic niche model for a candidate biofuel crop, Millettia pinnata, and apply the model to different climatic and irrigation scenarios to estimate the current and future patterns of climate suitability for its growth and naturalization. We use Australia as a case study for interpreting the niche model in terms that may be informative for both biofuels proponents and biosecurity regulators to plan management programmes that reflect the invasive potential in different areas. The model suggests that suitable growing conditions for M. pinnata in Australia are naturally restricted to the moist and semimoist tropics. Irrigation can extend the suitable growing conditions more widely throughout the tropics, and into more arid regions. Under future climate scenarios, suitable growing conditions for M. pinnata under natural rainfall contract towards the east coast, and extend southward into the subtropics. With irrigation, M. pinnata appears to have the potential in the future to naturalize across much of Australia. The bioclimatic modelling method demonstrated here is comparatively quick and easy, and can produce a rich array of data products to inform the interests of both bioenergy proponents and biosecurity regulators. We show how this modelling can support the development of spatially explicit biosecurity policies designed to manage invasion risks in a manner that balances bioenergy and biosecurity concerns.     

Acute Fluoxetine Treatment Induces Slow Rolling of Leukocytes on Endothelium in Mice

ObjectiveActivated platelets release serotonin at sites of inflammation where it acts as inflammatory mediator and enhances recruitment of neutrophils. Chronic treatment with selective serotonin reuptake inhibitors (SSRI) depletes the serotonin storage pool in platelets, leading to reduced leukocyte recruitment in murine experiments. Here, we examined the direct and acute effects of SSRI on leukocyte recruitment in murine peritonitis.MethodsC57Bl/6 and Tph1−/− (Tryptophan hydroxylase1) mice underwent acute treatment with the SSRI fluoxetine or vehicle. Serotonin concentrations were measured by ELISA. Leukocyte rolling and adhesion on endothelium was analyzed by intravital microscopy in mesentery venules with and without lipopolysaccharide challenge. Leukocyte extravasation in sterile peritonitis was measured by flow cytometry of abdominal lavage fluid.ResultsPlasma serotonin levels were elevated 2 hours after fluoxetine treatment (0.70±0.1 µg/ml versus 0.27±0.1, p = 0.03, n = 14), while serum serotonin did not change. Without further stimulation, acute fluoxetine treatment increased the number of rolling leukocytes (63±8 versus 165±17/0.04 mm²min⁻¹) and decreased their velocity (61±6 versus 28±1 µm/s, both p<0.0001, n = 10). In Tph1−/− mice leukocyte rolling was not significantly influenced by acute fluoxetine treatment. Stimulation with lipopolysaccharide decreased rolling velocity and induced leukocyte adhesion, which was enhanced after fluoxetine pretreatment (27±3 versus 36±2/0.04 mm², p = 0.008, n = 10). Leukocyte extravasation in sterile peritonitis, however, was not affected by acute fluoxetine treatment.ConclusionsAcute fluoxetine treatment increased plasma serotonin concentrations and promoted leukocyte-endothelial interactions in-vivo, suggesting that serotonin is a promoter of acute inflammation. E-selectin was upregulated on endothelial cells in the presence of serotonin, possibly explaining the observed increase in leukocyte-endothelial interactions. However transmigration of neutrophils in sterile peritonitis was not affected by higher serotonin concentrations, indicating that the effect of fluoxetine was restricted to early steps in the leukocyte recruitment. Whether SSRI use in humans alters leukocyte recruitment remains to be investigated.     

Rat erythrocyte glycophorins can be isolated by the lithium diiodosalicylate method used for other glycophorins.

1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrocytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiff's reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.     

Diversity loss in the macrophyte vegetation of northwest German streams and rivers between the 1950s and 2010

This resampling study in 338 semi-permanent plots analyses changes in river macrophyte diversity in 70 water courses (small streams to medium-sized rivers) from four regions of the northwest German lowlands during the last six decades. The total macrophyte species pool decreased between the 1950s and 2010/2011 by 28% (from 51 to 37 species), mean plot-level species richness by 19% (from 4.7 to 3.8 species per relevé) and the number of red-listed species by 40% (from 30 to 18 species). Species loss was associated with marked change in species traits: species with presumably higher mechanical stress tolerance (indicated by low specific leaf area and short leaf longevity) are more abundant today. Nearly, half of the species present in the 1950s had either disappeared or been replaced by other species in the recent relevés. The dramatic impoverishment is likely a consequence of continued nutrient input that drove oligo- and mesotraphent species to extinction, and of restructuring and maintenance works in the water courses that reduced stagnant and undisturbed river habitats, where stress-intolerant species can persist. Efficient measures to reduce the nutrient load and to re-naturalise stream and river beds are urgently needed to halt and reverse the loss of macrophyte diversity.     

Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen

Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α₂β₁, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen.