Characterization of the properties and trafficking of an anchorless form of the prion protein

Conversion of PrP(C) into PrP(Sc) is the central event in the pathogenesis of transmissible prion diseases. Although the molecular basis of this event and the intracellular compartment where it occurs are not yet understood, the association of PrP with cellular membranes and in particular its presence in detergent-resistant microdomains appears to be of critical importance. In addition it appears that scrapie conversion requires membrane-bound glycosylphosphatidylinositol (GPI)-linked PrP. The GPI anchor may affect either the conformation, the intracellular localization, or the association of the prion protein with specific membrane domains. However, how this occurs is not known. To understand the relevance of the GPI anchor for the cellular behavior of PrP, we have studied the biosynthesis and localization of a PrP version which lacks the GPI anchor attachment signal (PrP Delta GPI). We found that PrP Delta GPI is tethered to cell membranes and associates to membrane detergent-resistant microdomains but does not assume a transmembrane topology. Differently to PrP(C), this protein does not localize at the cell surface but is mainly released in the culture media in a fully glycosylated soluble form. The cellular behavior of anchorless PrP explains why PrP Delta GPI Tg mice can be infected but do not show the classical signs of the disorder, thus indicating that the plasma membrane localization of PrP(C) and/or of the converted scrapie form might be necessary for the development of a symptomatic disease

Analysis of General Practitioners’ Attitudes and Beliefs about Psychological Intervention and the Medicine-Psychology Relationship in Primary Care: Toward a New Comprehensive Approach to Primary Health Care

The biopsychosocial paradigm is a model of care that has been proposed in order to improve the effectiveness of health care by promoting collaboration between different professions and disciplines. However, its application still faces several issues. A quantitative-qualitative survey was conducted on a sample of general practitioners (GPs) from Milan, Italy, to investigate their attitudes and beliefs regarding the role of the psychologist, the approach adopted to manage psychological diseases, and their experiences of collaboration with psychologists. The results show a partial view of the psychologist’s profession that limits the potential of integration between medicine and psychology in primary care. GPs recognized that many patients (66%) would often benefit from psychological intervention, but only in a few cases (9%) were these patients regularly referred to a psychologist. Furthermore, the referral represents an almost exclusive form of collaboration present in the opinions of GPs. Only 8% of GPs would consider the joint and integrated work of the psychologist and doctor useful within the primary health care setting. This vision of the role of psychologists among GPs represents a constraint in implementing a comprehensive primary health care approach, as advocated by the World Health Organization

The use of virtual environments for survey spatial ability evaluation in topographical disorientation

Due to their interactivity and to the sense of presence they afford, virtual environments constitute an interesting opportunity to study spatial cognition. In accordance with this perspective, we aimed to introduce a spatial test in virtual simulation in order to investigate the survey spatial ability in patients with topographical disorientation. To do this, we used the “planning in advance task” in a virtual environment that constitutes an effective procedure to experimentally evaluate survey maps. With this procedure we present the single case of a woman, with a right medial temporal lobe lesion, who shows a selective impairment in the acquisition of new spatial relationships. The patient’s performance in “planning in advance task” was compared with that of a control group made up of 40 female subjects matched for age and education. Results show how the patient revealed a significantly lower spatial performance when compared to the control group, demonstrating an inability to solve survey-type spatial tasks in complex virtual environments

Health care: social "anchorage" construction (a qualitative approach)

En el presente trabajo, que es parte de un proyecto mayor de investigación, destacamos la importancia de indagar las representaciones sociales de cuidado de la salud y sobre las diferentes prácticas asociadas a ellas con el fin de analizar el "anclaje" social de su construcción. Consideramos que el papel que tiene la cultura en el comportamiento humano es un factor relevante en la adopción de una modalidad de cuidado de la salud por parte de los sujetos sociales. En el marco del análisis teórico del binomio cuidado de la salud-cultura observamos tres aspectos de importancia: 1- que los comportamientos están arraigados en creencias y tradiciones culturales complejas, 2- que expresan paradojas comportamentales y ,3-que, en el caso del presente estudio en particular, están ligados a estereotipos de género. Aportamos material empírico obtenido a través de la aplicación de entrevistas semiestructuradas en una prueba piloto sobre una muestra de mujeres de la ciudad de La Plata, Argentina.As a part of a mayor research this study outlines the concern of investigate social representations regarding care health and the "anchorage" process of construction. We believe that the paper of the culture is decisive at the time to adopt an style of health care. Theoretical analysis about culture- health care implies three issues: 1- Behaviours are emboiled in cultural scripts, 2- Health-care reveals behaviourals paradoxes, 3- Gender perspective. Empirical data were obtain from a pilot sample of women all of which residents in La Plata, Argentina. The personal interview was modeled on the semiestructured clinical interview.Trabajo realizado para el Proyecto de Investigación Programa de Incentivos UNLP 2002-2004 "Educación sexual: demandas sociales de cuidado de la salud y sus espacios de representación", dirigido por la Dra. Ana Candreva.Facultad de Ciencias Médica

Mitochondrial Abnormalities in Down Syndrome: Pathogenesis, Effects and Therapeutic Approaches

Down syndrome (DS) consists of a complex phenotype with constant features, such as mental retardation and hypotonia, and variable features, including heart defects and susceptibility to Alzheimer’s disease, type 2 diabetes, obesity and immune disorders. Overexpression of genes mapping to chromosome 21 (Hsa21) is directly or indirectly responsible for pathogenesis of DS phenotypic features, as overexpressed Hsa21 genes dysregulate several other genes mapping to different chromosomes. Many of these genes are involved in mitochondrial function. Recent studies highlight a link between mitochondrial dysfunction, consistently observed in DS subjects, and DS phenotype. In this review, we first provide a basic overview of mitochondrial alterations in DS in terms of mitochondrial bioenergetics, biogenesis and morphology. We then discuss how mitochondrial malfunction may contribute to the pathogenesis of clinical manifestations and how specific Hsa21 genes may cause the disruption of mitochondrial phenotype. Finally, we focus on drugs, which affect mitochondrial function and network to propose possible therapeutic approaches aimed at improving and/or preventing various aspects of the DS phenotype. Our working hypothesis is that correcting the mitochondrial defect might improve the neurological phenotype and prevent DS-associated pathologies, thus providing a better quality of life for DS individuals and their families

Large-scale comparative analysis of cytogenetic markers across Lepidoptera

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

The cellular prion protein (PrP(C)) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP(Sc)). Although endocytosis appears to be required for this conversion, the mechanism of PrP(C) internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the mechanism of PrP(C) endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP(C) internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP(C) endocytosis. PrP(C) internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP(C) co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP(C) can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. CONCLUSIONS/SIGNIFICANCE: These findings are of particular interest if we consider that the internalization route/s undertaken by PrP(C) can be crucial for the ability of different prion strains to infect and to replicate in different cell lines