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

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

Visualization and Identification of IL-7 Producing Cells in Reporter Mice

Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging

Visualization and Identification of IL-7 Producing Cells in Reporter Mice

Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging

Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA

CD8+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens

Insight into the Mechanisms of Adenovirus Capsid Disassembly from Studies of Defensin Neutralization

Defensins are effectors of the innate immune response with potent antibacterial activity. Their role in antiviral immunity, particularly for non-enveloped viruses, is poorly understood. We recently found that human alpha-defensins inhibit human adenovirus (HAdV) by preventing virus uncoating and release of the endosomalytic protein VI during cell entry. Consequently, AdV remains trapped in the endosomal/lysosomal pathway rather than trafficking to the nucleus. To gain insight into the mechanism of defensin-mediated neutralization, we analyzed the specificity of the AdV-defensin interaction. Sensitivity to alpha-defensin neutralization is a common feature of HAdV species A, B1, B2, C, and E, whereas species D and F are resistant. Thousands of defensin molecules bind with low micromolar affinity to a sensitive serotype, but only a low level of binding is observed to resistant serotypes. Neutralization is dependent upon a correctly folded defensin molecule, suggesting that specific molecular interactions occur with the virion. CryoEM structural studies and protein sequence analysis led to a hypothesis that neutralization determinants are located in a region spanning the fiber and penton base proteins. This model was supported by infectivity studies using virus chimeras comprised of capsid proteins from sensitive and resistant serotypes. These findings suggest a mechanism in which defensin binding to critical sites on the AdV capsid prevents vertex removal and thereby blocks subsequent steps in uncoating that are required for release of protein VI and endosomalysis during infection. In addition to informing the mechanism of defensin-mediated neutralization of a non-enveloped virus, these studies provide insight into the mechanism of AdV uncoating and suggest new strategies to disrupt this process and inhibit infection

TNFAIP3 Maintains Intestinal Barrier Function and Supports Epithelial Cell Tight Junctions

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3−/− mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3−/− mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation

Polarized Secretion of Drosophila EGFR Ligand from Photoreceptor Neurons Is Controlled by ER Localization of the Ligand-Processing Machinery

Trafficking within the endoplasmic reticulum and specialized localization of the intra-membrane protease Rhomboid regulate EGF ligand-dependent signaling in Drosophila photoreceptor axon termini