A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C

Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context

Reconceiving Barriers for Democratic Health Education in Danish Schools: an Analysis of Institutional Rationales

Health promotion - and education researchers and practitioners advocate for more democratic approaches to school-based health education, including participatory teaching methods and the promotion of a broad and positive concept of health and health knowledge, including aspects of the German educational concept of bildung. Although Denmark, from where the data of this article are derived, has instituted policies for such approaches, their implementation in practice faces challenges. Adopting a symbolic interactionist analytical framework this paper explores and defines two powerful institutional rationales connected to formal and informal social processes and institutional purposes of schools, namely conservatism and Neoliberalism. It is empirically described and argued how these institutional rationales discourage teachers and students from including a broad and positive concept of health, the element of participation, and the promotion of general knowledge as legitimate elements in health education. This paper thus contains a perspective on health education practice, which, in a new way, contributes to explain the relatively slow progress of democratic approaches to school health education

Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer

<p>Abstract</p> <p>Background</p> <p>LIM kinase 1 (LIMK1), a LIM domain containing serine/threonine kinase, modulates actin dynamics through inactivation of the actin depolymerizing protein cofilin. Recent studies have indicated an important role of LIMK1 in growth and invasion of prostate and breast cancer cells; however, the molecular mechanism whereby LIMK1 induces tumor progression is unknown. In this study, we investigated the effects of ectopic expression of LIMK1 on cellular morphology, cell cycle progression and expression profile of LIMK1 in prostate tumors.</p> <p>Results</p> <p>Ectopic expression of LIMK1 in benign prostatic hyperplasia cells (BPH), which naturally express low levels of LIMK1, resulted in appearance of abnormal mitotic spindles, multiple centrosomes and smaller chromosomal masses. Furthermore, a transient G1/S phase arrest and delayed G2/M progression was observed in BPH cells expressing LIMK1. When treated with chemotherapeutic agent Taxol, no metaphase arrest was noted in these cells. We have also noted increased nuclear staining of LIMK1 in tumors with higher Gleason Scores and incidence of metastasis.</p> <p>Conclusion</p> <p>Our results show that increased expression of LIMK1 results in chromosomal abnormalities, aberrant cell cycle progression and alteration of normal cellular response to microtubule stabilizing agent Taxol; and that LIMK1 expression may be associated with cancerous phenotype of the prostate.</p

Gender specific age-related changes in bone density, muscle strength and functional performance in the elderly: a-10 year prospective population-based study

Background:&nbsp;Age-related losses in bone mineral density (BMD), muscle strength, balance, and gait have been linked to&nbsp;an increased risk of falls, fractures and disability, but few prospective studies have compared the timing, rate and pattern&nbsp;of changes in each of these measures in middle-aged and older men and women. This is important so that targeted&nbsp;strategies can be developed to optimise specific musculoskeletal and functional performance measures in older adults.&nbsp;Thus, the aim of this 10-year prospective study was to: 1) characterize and compare age- and gender-specific changes in&nbsp;BMD, grip strength, balance and gait in adults aged 50 years and over, and 2) compare the relative rates of changes&nbsp;between each of these musculoskeletal and functional parameters with ageing.Methods: Men (n = 152) and women (n = 206) aged 50, 60, 70 and 80 years recruited for a population-based study had&nbsp;forearm BMD, grip strength, balance and gait velocity re-assessed after 10-years.Results: The annual loss in BMD was 0.5-0.7% greater in women compared to men aged 60 years and older&nbsp;(p &lt; 0.05- &lt; 0.001), but there were no gender differences in the rate of loss in grip strength, balance or gait. From the age&nbsp;of 50 years there was a consistent pattern of loss in grip strength, while the greatest deterioration in balance and gait&nbsp;occurred from 60 and 70 years onwards, respectively. Comparison of the changes between the different measures&nbsp;revealed that the annual loss in grip strength in men and women aged &lt;70 years was 1-3% greater than the decline in&nbsp;BMD, balance and gait velocity.Conclusion: There were no gender differences in the timing (age) and rate (magnitude) of decline in grip strength,&nbsp;balance or gait in Swedish adults aged 50 years and older, but forearm BMD decreased at a greater rate in women than&nbsp;in men. Furthermore, there was heterogeneity in the rate of loss between the different musculoskeletal and function&nbsp;parameters, especially prior to the age of 70 years, with grip strength deteriorating at a greater rate than BMD,&nbsp;balance and gait.</div

Deletion of the Human Cytomegalovirus US17 Gene Increases the Ratio of Genomes per Infectious Unit and Alters Regulation of Immune and Endoplasmic Reticulum Stress Response Genes at Early and Late Times after Infection

Human cytomegalovirus (HCMV) employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves capture of a host gene and then its successive duplication and divergence, forming a family of genes, many of which have immunomodulatory activities. The HCMV US12 family consists of 10 tandemly arranged sequence-related genes in the unique short (US) region of the HCMV genome (US12 to US21). Each gene encodes a protein possessing seven predicted transmembrane domains, patches of sequence similarity with cellular G-protein-coupled receptors, and the Bax inhibitor 1 family of antiapoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray analysis of cells infected with a recombinant HCMV isolate with a US17 deletion (the ΔUS17 mutant virus) revealed blunted host innate and interferon responses at early times after infection (12 h postinfection [hpi]), a pattern opposite that previously seen in the absence of the immunomodulatory tegument protein pp65 (pUL83). Although the ΔUS17 mutant virus produced numbers of infectious particles in fibroblasts equal to the numbers produced by the parental virus, it produced \u3e3-fold more genome-containing noninfectious viral particles and delivered increased amounts of pp65 to newly infected cells. These results suggest that US17 has evolved to control virion composition, to elicit an appropriately balanced host immune response. At later time points (96 hpi), ΔUS17 mutant-infected cells displayed aberrant expression of several host endoplasmic reticulum stress response genes and chaperones, some of which are important for the final stages of virion assembly and egress. Our results suggest that US17 modulates host pathways to enable production of virions that elicit an appropriately balanced host immune response