OSTM1 regulates beta-catenin/Lef1 interaction and is required for Wnt/beta-catenin signaling

The Wnt/beta-catenin signaling pathway controls key aspects of embryonic development and adult tissue homeostasis, including the formation and maintenance of bone. Recently, mutations in the OSTM1 gene were found to be the cause of severe autosomal recessive osteopetrosis in both the mouse and humans. This disorder is characterized by increased bone mass resulting from a defect in osteoclast maturation. The possible role of OSTM1 in signaling of the Wnt/beta-catenin "canonical" pathway was investigated in totipotent mouse F9 embryonal teratocarcinoma cells. Overexpression of OSTM1 in F9 cells increased Wnt3a-responsive beta-catenin accumulation and Lef/Tcf-sensitive transcription. Similarly, knockdown of endogenous OSTM1 attenuated the ability of Wnt3a to stimulate the canonical signaling pathway. An OSTM1 mutant (detected in humans with osteopetrosis) was expressed in F9 cells and found to inhibit Wnt-stimulated beta-catenin stabilization, gene transcription, and primitive endoderm formation. Expression of this OSTM1 C-terminal deletion mutant attenuated Lef/Tcf-sensitive gene transcription, even when transcription was activated by expression of a constitutively-active form of beta-catenin. However, expression of this OSTM1 C-terminal deletion mutant was unable to alter Lef/Tcf-sensitive gene transcription when transcription was activated by expression of a beta-catenin/Lef chimeric protein. From the standpoint of protein-protein interactions, expression of wild-type OSTM1 stimulated whereas mutant OSTM1 inhibited, the Wnt-dependent association of beta-catenin and Lef1. On the foundation of these experiments, we propose that the human mutations in OSTM1 such as the C-terminal deletion mutant studied herein provoke dysregulation of the canonical Wnt/beta-catenin signaling pathway, providing a molecular basis for severe autosomal recessive osteopetrosis

Comparison of porcine thorax to gelatine blocks for wound

Published online first in International Journal of Legal Medicine. The support of EPSRC and The Home Office are recognised. Open Access, this article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:/ /creativecommons.org/licenses/by/4.0/)Tissue simulants are typically used in ballistic testing as substitutes for biological tissues. Many simulants have been used, with gelatine amongst the most common. While two concentrations of gelatine (10 and 20 %) have been used extensively, no agreed standard exists for the preparation of either. Comparison of ballistic damage produced in both concentrations is lacking. The damage produced in gelatine is also questioned, with regards to what it would mean for specific areas of living tissue. The aim of the work discussed in this paper was to consider how damage caused by selected pistol and rifle ammunition varied in different simulants. Damage to gelatine blocks 10 and 20 % in concentration were tested with 9 mm Luger (9 × 19 full metal jacket; FMJ) rounds, while damage produced by .223 Remington (5.56 × 45 Federal Premium® Tactical® Bonded®) rounds to porcine thorax sections (skin, underlying tissue, ribs, lungs, ribs, underlying tissue, skin; backed by a block of 10 % gelatine) were compared to 10 and 20 % gelatine blocks. Results from the .223 Remington rifle round, which is one that typically expands on impact, revealed depths of penetration in the thorax arrangement were significantly different to 20 % gelatine, but not 10 % gelatine. The level of damage produced in the simulated thoraxes was smaller in scale to that witnessed in both gelatine concentrations,though greater debris was produced in the thoraxes.The support of EPSRC and The Home Office are recognised

Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking

BACKGROUND: Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta (2)-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta(2)-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase. RESULTS: The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta(2)-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta(2)-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β(2)-adrenergic receptor for those of Ste2 creates a Ste2/beta(2)-adrenergic receptor chimera displaying insulin-stimulated internalization. CONCLUSION: Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins