The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

The differentiation and senescence programs of metazoans play key roles in regulating normal development and preventing aberrant cell proliferation, such as cancer. These programs are intimately associated with both the mitotic and apoptotic pathways. Caspase-8 is an apical apoptotic initiator that has recently been appreciated to coordinate non-apoptotic roles in the cell. Most of these functions are attributed to the catalytic domain, however, the amino-terminal death effector domains (DED)s, which belong to the death domain superfamily of proteins, can also play key roles during development. Here we describe a novel role for caspase-8 DEDs in regulating cell differentiation and senescence. Caspase-8 DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial and myeloid cells; genetic deletion or shRNA suppression of caspase-8 disrupts cell differentiation, while re-expression of DEDs rescues this phenotype. Among caspase-8 deficient neuroblastoma cells, DED expression attenuated tumor growth in vivo and proliferation in vitro via disruption of mitosis and cytokinesis, resulting in upregulation of p53 and induction of differentiation markers. These events occur independent of caspase-8 catalytic activity, but require a critical lysine (K156) in a microtubule-binding motif in the second DED domain. The results demonstrate a new function for the DEDs of caspase-8, and describe an unexpected mechanism that contributes to cell differentiation and senescence

Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

<p>Abstract</p> <p>Background</p> <p>β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured <it>in vitro </it>using His-tag purified proteins and <it>in vivo </it>in a β-carotene-accumulating <it>E. coli </it>system.</p> <p>Results</p> <p>Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our <it>ab initio </it>model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.</p> <p>Conclusions</p> <p>Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.</p

Organizing risk: organization and management theory for the risk society

Risk has become a crucial part of organizing, affecting a wide range of organizations in all sectors. We identify, review and integrate diverse literatures relevant to organizing risk, building on an existing framework that describes how risk is organized in three ‘modes’ – prospectively, in real-time, and retrospectively. We then identify three critical issues in the existing literature: its fragmented nature; its neglect of the tensions associated with each of the modes; and its tendency to assume that the meaning of an object in relation to risk is singular and stable. We provide a series of new insights with regard to each of these issues. First, we develop the concept of a risk cycle that shows how organizations engage with all three modes and transition between them over time. Second, we explain why the tensions have been largely ignored and show how studies using a risk work perspective can provide further insights into them. Third, we develop the concept of risk translation to highlight the ways in the meanings of risks can be transformed and to identify the political consequences of such translations. We conclude the paper with a research agenda to elaborate these insights and ideas further

The Sooty show laid bear - Children, puppets and make-believe

This article seeks to examine the complex nature of pretence as portrayed in a popular UK children's television puppet show. Central to the various theoretical concerns with make-believe philosophical, psychological and lucid - has been the phenomenological axis of the 'real-imaginary'. Because of the serial types of transformations undertaken by characters in the programme and the reliance placed on natural models of conversation, the problematic nature of this core boundary is highlighted. Despite its overt status as a fictional representation of animal behaviour, the very animality of the puppets is rendered opaque as their identities as children are linguistically accomplished. As a consequence we argue that the show as a piece of representational art is structured by moral and behavioural dictates typical of conventional adult-child interaction