A Tail-Anchored Myotonic Dystrophy Protein Kinase Isoform Induces Perinuclear Clustering of Mitochondria, Autophagy, and Apoptosis

Contains fulltext : 79678.pdf (publisher's version ) (Open Access)BACKGROUND: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. CONCLUSION/SIGNIFICANCE: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients

ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions

Modular actin nano-architecture enables podosome protrusion and mechanosensing

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries

Inherited determinants of Crohn's disease and ulcerative colitis phenotypes: a genetic association study

Crohn's disease and ulcerative colitis are the two major forms of inflammatory bowel disease; treatment strategies have historically been determined by this binary categorisation. Genetic studies have identified 163 susceptibility loci for inflammatory bowel disease, mostly shared between Crohn's disease and ulcerative colitis. We undertook the largest genotype association study, to date, in widely used clinical subphenotypes of inflammatory bowel disease with the goal of further understanding the biological relations between diseases

Sell-side analysts' use and communication of intellectual capital information

Structural economic changes in many countries, together with unprecedented developments in the business environment, have significantly affected the value creation processes of firms and the way business is conducted. The traditional financial reporting model is inadequate as a consequence of these developments, and intellectual capital (IC) information has gained importance for investment decision making. Empirical capital markets research demonstrates the value-relevance and predictive ability of certain types of IC information. The use of IC information by capital market participants is a topic that has begun to gain attention from contemporary researchers, but for which scant empirical evidence exists. Much of the research in this area relies on the literature about the use of non-financial information (NFI), which is inadequate in its examination of certain types of IC information. Therefore, the main aim of this thesis is to examine the use and communication of IC information by sell-side analysts. Sell-side analysts are of particular interest because they are capital market intermediaries and sophisticated processors of corporate information. The reports they produce provide an opportunity to examine their use and communication of IC information. The specific objectives of this thesis are to examine: the extent and types of IC information used by sell-side analysts in initiating coverage reports produced by them; how IC information is used and communicated in these reports; and factors that may influence the use of IC information by sell-side analysts. In order to address these research objectives a content analysis of IC references in 64 initiating coverage reports written on an equivalent number of S&P/ASX 200/300 companies is performed. The content analysis identifies and measures IC references by topic, evidence (discursive, monetary, numerical, or visual), news-tenor (positive, neutral or negative) and time orientation (forward-looking, past-oriented or non-time-specific). The findings indicate that Australian sell-side analysts appreciate the importance of IC in firm valuation, and thus are not ambivalent about the use of IC information in general. However, the findings suggest that their communication of IC information is inconsistent and unsystematic, and inadequate in relation to certain types of IC. This highlights the need for undertaking work at a policy level to educate and train sell-side analysts to deal with IC information, and the development of better models and guidelines for analysing and communicating IC information. On how IC information is used, this thesis finds that sell-side analysts have varying uses of IC information. It was found that IC is predominantly communicated discursively, positively, and in a past-oriented manner; and in doing so IC is used as a tool to further the sell-side analysts’ agenda for the company analysed. Further, the results highlight that the type of investment recommendation in analyst reports impacts on the evidence, news tenor, and time-orientation of IC communicated. These findings alert future researchers to the wider role played by IC beyond its use in forecasts and valuations. Also, the findings indicate inter-sectoral differences in the use of IC information in analyst reports, highlighting the need to improve IC reporting practices of firms by including additional information on industry-specific IC value drivers. Further, it was found that sell-side analysts emphasise IC information in analyst reports for companies from high IC-intensive sectors compared to those from low IC-intensive sectors. Similarly, it was found that analyst reports on risky companies contain significantly more IC information than analyst reports on less risky companies. Contrary to expectations, the extent of IC information is not found to vary with firm size and firm profitability. Also, the results support that the extent of certain types of IC information differs between types of analysts’ investment recommendations. More generally, the findings of this thesis suggest that the corporate reporting process could be improved by including additional types of IC information and providing this information more effectively in a manner that enables users to visualise the interrelationships between resources (both tangible and intangible) and outcomes. This study calls for standards or guidelines for intellectual capital reporting (ICR) in Australia and the expansion of the role of auditing and assurance services to enhance reliability of firm provided IC information in a bid to improve the use of IC information in company analysis by sell-side analysts

Identification of a Rat Model for Usher Syndrome Type 1B by N-Ethyl-N-nitrosourea Mutagenesis-Driven Forward Genetics

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat