ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.     

Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina

Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.     

Thermoreversible protein hydrogel as cell scaffold

A thermoreversible fibrillar hydrogel has been formed from an aqueous lysozyme solution in the presence of dithiothreitol (DTT). Its physical properties and potential as a tissue engineering scaffold have been explored. Hydrogels were prepared by dissolving 3 mM protein in a 20 mM DTT/water mixture, heating to 85 degrees C and cooling at room temperature. No gel was observed for the equivalent sample without DTT. The elastic nature of the gel formed was confirmed by rheology, and the storage modulus of our gel was found to be of the same order of magnitude as for other cross-linked biopolymers. Micro differential scanning calorimetry (microDSC) experiments confirmed that the hydrogel was thermally reversible and that gelation and melting occurs through a solid-liquid-like first-order transition. Infrared spectroscopy of the hydrogel and transmission electron microscopy studies of very dilute samples revealed the presence of beta-sheet-rich fibrils that were approximately 4-6 nm in diameter and 1 mum in length. These fibrils are thought to self-assemble along their long axes to form larger fibers that become physically entangled to form the three-dimensional network observed in both cryo-scanning electron microscopy (cryo-SEM) and small-angle neutron scattering (SANS) studies. The hydrogel was subsequently cultured with 3T3 fibroblasts and cells spread extensively after 7 days and stretched actin filaments formed that were roughly parallel to each other, indicating the development of organized actin filaments in the form of stress fibers in cells.     

The role of temperature in determining distributions and coexistence of three species of Plestiodon

The Common Five-Lined Skink (Plestiodon fasciatus), Southeastern Five-Lined Skink (P. inexpectatus), and Broadheaded Skink (P. laticeps) are all found in forested ecosystems of the southeastern United States and occur in apparent sympatry. No aspect of their ecology has been quantitatively shown to be different. Differences in their natural history, such as northern extent of geographic range, observations of differences in habitat preference, and differing physiological responses to temperature, implicate the thermal environment as a potential niche dimension by which these organisms partition resources. Here we show that the northernmost extent of each species' range can be adequately discriminated by temperature extremes and that their preferred habitats differ in canopy cover, mean temperature, and daily maximum temperatures. These differences are supported by observations in the field. Based upon these findings and observations, we conclude that these species may be able to exist in such close proximity due to differences in their thermal niche, particularly the two like-sized species, P. fasciatus and P. inexpectatus. P. laticeps is a much larger lizard as an adult and functionally coexists with its congeners, potentially by partitioning resources along a different axis.     

Evolutionarily consistent families in SCOP: sequence, structure and function

Background

HutZ is the sole heme storage protein identified in the pathogenic bacterium Vibrio cholerae and is required for optimal heme utilization. However, no heme oxygenase activity has been observed with this protein. Thus far, HutZ??s structure and heme-binding mechanism are unknown.

Results

We report the first crystal structure of HutZ in a homodimer determined at 2.0 ? resolution. The HutZ structure adopted a typical split-barrel fold. Through a docking study and site-directed mutagenesis, a heme-binding model for the HutZ dimer is proposed. Very interestingly, structural superimposition of HutZ and its homologous protein HugZ, a heme oxygenase from Helicobacter pylori, exhibited a structural mismatch of one amino acid residue in ??6 of HutZ, although residues involved in this region are highly conserved in both proteins. Derived homologous models of different single point variants with model evaluations suggested that Pro¹⁴⁰ of HutZ, corresponding to Phe²¹⁵ of HugZ, might have been the main contributor to the structural mismatch. This mismatch initiates more divergent structural characteristics towards their C-terminal regions, which are essential features for the heme-binding of HugZ as a heme oxygenase.

Conclusions

HutZ??s deficiency in heme oxygenase activity might derive from its residue shift relative to the heme oxygenase HugZ. This residue shift also emphasized a limitation of the traditional template selection criterion for homology modeling.     

H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.     

Prediction of Protein-Destabilizing Polymorphisms by Manual Curation with Protein Structure

The relationship between sequence polymorphisms and human disease has been studied mostly in terms of effects of single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions that change protein structure and function. However, less attention has been paid to more drastic sequence polymorphisms which cause premature termination of a protein’s sequence or large changes, insertions, or deletions in the sequence. We have analyzed a large set (n = 512) of insertions and deletions (indels) and single nucleotide polymorphisms causing premature termination of translation in disease-related genes. Prediction of protein-destabilization effects was performed by graphical presentation of the locations of polymorphisms in the protein structure, using the Genomes TO Protein (GTOP) database, and manual annotation with a set of specific criteria. Protein-destabilization was predicted for 44.4% of the nonsense SNPs, 32.4% of the frameshifting indels, and 9.1% of the non-frameshifting indels. A prediction of nonsense-mediated decay allowed to infer which truncated proteins would actually be translated as defective proteins. These cases included the proteins linked to diseases inherited dominantly, suggesting a relation between these diseases and toxic aggregation. Our approach would be useful in identifying potentially aggregation-inducing polymorphisms that may have pathological effects.     

A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses

Understanding the mechanisms by which pathogens induce vascular inflammation and dysfunction may reveal novel therapeutic targets in sepsis and related conditions. The intracellular receptor NOD1 recognises peptidoglycan which features in the cell wall of Gram negative and some Gram positive bacteria. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways. We have previously shown that stimulation of NOD1 directly activates blood vessels and causes experimental shock in vivo. In this study we have used an ex vivo vessel-organ culture model to characterise the relative contribution of the endothelium in the response of blood vessels to NOD1 agonists. In addition we present the novel finding that NOD1 directly activates human blood vessels. Using human cultured cells we confirm that endothelial cells respond more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This paper is the first to demonstrate activation of whole human artery by NOD1 stimulation and the relative importance of the endothelium in the sensing of NOD1 ligands by vessels. This data supports the potential utility of NOD1 and RIP2 as therapeutic targets in human disease where vascular inflammation is a clinical feature, such as in sepsis and septic shock.     

Phenotyping for Abiotic Stress Tolerance in Maize

The ability to quickly develop germplasm having tolerance to several complex polygenic inherited abiotic and biotic stresses combined is critical to the resilience of cropping systems in the face of climate change.Molecular breeding offers the tools to accelerate cereal breeding;however,suitable phenotyping proto-cols are essential to ensure that the much-anticipated benefits of molecular breeding can be realized.To facilitate the full potential of molecular tools,greater emphasis needs to be given to reducing the within-experimental site variability,application of stress and characterization of the environment and appropriate phenotyping tools.Yield is a function of many processes throughout the plantcycle,and thus integrative traits that encompass crop performance over time or organization level(i.e.canopy level) will provide a better alternative to instantaneous measurements which provide only a snapshot of a given plant process.Many new phenotyping tools based on remote sensing are now available including non-destructive measurements of growth-related parameters based on spectral reflectance and infrared thermometry to estimate plant water status.Here we describe key field phenotyping protocols for maize with emphasis on tolerance to drought and low nitrogen.