Uwe Dressel

Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily

ABSTRACT Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor’s silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathioneS-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.

Vitamin D action and regulation of bone remodeling : suppression of osteoclastogenesis by the mature osteoblast

Vitamin D acts through the immature osteoblast to stimulate osteoclastogenesis. Transgenic elevation of VDR in mature osteoblasts was found to inhibit osteoclastogenesis associated with an altered OPG response. This inhibition was confined to cancellous bone. This study indicates that vitamin D–mediated osteoclastogenesis is regulated locally by OPG production in the mature osteoblast.

Not a minute to waste

We are finally beginning to unlock the mechanisms underlying Ca2+-stimulated muscle differentiation and cytokine-mediated muscle wasting. Gaining a better understanding of the signaling pathways that regulate muscle development and decay improves the prospects for repairing aged, injured and diseased muscle.

A genetic screen for impaired systemic RNAi highlights the crucial role of Dicer-like 2

DCL2 is required for efficient RDR6-dependent systemic posttranslational gene silencing, recruiting RNA-DEPENDENT RNA POLYMERASE6 and promoting the production of dsRNA that is mainly processed into 21-nucleotide siRNAs by DCL4 in wild-type plants. Posttranscriptional gene silencing (PTGS) of transgenes involves abundant 21-nucleotide small interfering RNAs (siRNAs) and low-abundance 22-nucleotide siRNAs produced from double-stranded RNA (dsRNA) by DCL4 and DCL2, respectively. However, DCL2 facilitates the recruitment of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to ARGONAUTE 1-derived cleavage products, resulting in more efficient amplification of secondary and transitive dsRNA and siRNAs. Here, we describe a reporter system where RDR6-dependent PTGS is initiated by restricted expression of an inverted-repeat dsRNA specifically in the Arabidopsis (Arabidopsis thaliana) root tip, allowing a genetic screen to identify mutants impaired in RDR6-dependent systemic PTGS. Our screen identified dcl2 but not dcl4 mutants. Moreover, grafting experiments showed that DCL2, but not DCL4, is required in both the source rootstock and the recipient shoot tissue for efficient RDR6-dependent systemic PTGS. Furthermore, dcl4 rootstocks produced more DCL2-dependent 22-nucleotide siRNAs than the wild type and showed enhanced systemic movement of PTGS to grafted shoots. Thus, along with its role in recruiting RDR6 for further amplification of PTGS, DCL2 is crucial for RDR6-dependent systemic PTGS.

Protein-protein cross-linking in the use of the eukaryotic eGST-fusion system

We describe here an unusual phenomenon in the isolation of protein complexes from eukaryotic cells using expressed GST-fusion proteins. Protein complexes are involved in a large number of regulatory mechanisms. Therefore, the use of tagged fusion proteins is an important tool for isolation of such protein complexes. For this purpose, we used the nuclear factor Alien, described as a corepressor for the thyroid hormone receptor, fused to the eukaryotic eGST and expressed this fusion in human cells. After affinity purification over glutathione-Sepharose using stringent washing steps, we observed several co-purifying bands migrating at molecular weights higher than the GST-Alien fusion protein. These bands appeared specifically in the GST-Alien transfected cell preparations. Surprisingly, using both Western blotting and MALDI-analyses, we revealed that these bands are composed of the GST-Alien protein itself. We hypothesize that overexpressed factors may generate unexpected cross-linking products which can confound the analyses of such affinity-purified complexes. The cross-linking products could not be eliminated by using beta-mercaptoethanol in the gel system and by boiling in SDS-sample buffer. Also, we demonstrate that Western blotting analyses using antibodies directed against both the tag-epitope and the expressed protein of interest can rapidly, reliably, and in a cost-saving manner identify such artifacts, eliminating them from the analyses of potentially interesting interaction partners. Our findings clearly show that the overexpression and purification of proteins from eukaryotic cells may generate unusual structural features that strongly influence complex formation and the migration in SDS-PAGE.

Agency theory perspective on public-private-partnerships: international development project

Purpose Agency theory suggests that divergences will occur when a principal, e.g. client, and agent e.g. a project manager, interests are different in the execution of a project. The purpose of this paper is to explore if the agency theory can explain the subtleties integral to the behaviours and relationships between players delivering a public-private-partnership (PPP) in the context of an international development (ID) project. The intra-/interpersonal dynamics include governments, non-governmental organisations (NGOs) and private commercial service providers. The authors develop a conceptual framework and provide evidence from a case study of the testing of a Road Safety Toolkit in Kenya to explore several propositions. Design/methodology/approach Extant literature identified application of the agency theory, and the development of a conceptual framework. A case study describing an ID project was used to validate the propositions prior to the expansion of a research instrument for data collection in the field. Findings Through the lens of the agency theory and the limitations imposed by exploring a series of propositions, several insightful conclusions have been derived from the case. ID projects have particular nuisances that make them unique when compared to the majority of commercial applications. An added dimension and level of complexity is a consequence of the PPP incorporating government, NGOs and private corporations. The case exemplified the need for PPP ID projects to build on partner networks to influence and disseminate outcomes. Some agency problems were far less prominent than would normally be seen in a commercial project. Research limitations/implications The methodologies presented in this paper need to be adapted and practiced in different kinds of ID projects in order to get confirmatory analytical results. The limitations imposed by the use of the single case, whilst drawing insightful conclusions, would necessitate greater testing in the field. Practical implications Although the problems of the agency theory are well researched in the operations management literature, there is limited application to ID projects and no previous research within the context of a PPP. Therefore, this work is important for greater understanding of the specific issues associated with project delivery of an ID. Social implications Conflicting goals between principals and agents are common for organisations, which in turn affect inter-relationships on an international footing. The agency theory has had little attention in the project management field, yet is fundamental to relationships and communication. Originality/value There has been little research that explores the agency theory in the context of a PPP involving governments, NGOs and private commercial service providers, executed as an ID project. This work, therefore, exhibits new and novel findings.

Chapter 3 PPARδ: Emerging therapeutic potential of novel agonists in lipid and glucose homeostasis

Publisher Summary PPARδ was first cloned in Xenopus and termed PPARβ. However, when first cloned in mammals, it was not obviously homologous to the Xenopus gene and given the name PPARδ. Other synonyms for PPARδ are NUC1 or FAAR. PPARδ is expressed in various tissues, often at a higher level than PPARα or PPARγ, where it activates genes that increase lipid absorption, preferential lipid utilization, lipid catabolism and β-oxidation, and energy expenditure. This chapter gives an overview on the function of PPARδ in metabolism and focuses on the molecular regulation of lipid and energy homeostasis modulated by agonists for this receptor and their therapeutic potentials. The chapter summarizes striking evidence that specific agonists for PPARδ ameliorate cholesterol homeostasis, have great potential in treating hyperlipidemia, cardiovascular disease, hypertension, and other metabolic diseases associated with obesity and the metabolic syndrome. Activation of PPARδ in skeletal muscle results in remodeling of this major peripheral mass tissue, increasing its oxidative fibers, and thereby contributing to the reduction of overall body-fat content. Dyslipidemia, hyperinsulinemia, blood glucose levels (and glucose tolerance), diabetes, inflammation, and obesity are all serious risk factors for cardiovascular and metabolic disease and PPARδ has been reported to ameliorate many of these metabolic perturbations.