1,720,955 research outputs found
Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance
No full textThe Semliki-Forest virus (SFV) system was used to overexpress human wild-type and mutant prion proteins as well as FLAG-tagged human and bovine PrP in mammalian cells. The application of recombinant SFV vectors allowed a high-level production of highly glycosylated prion proteins with a molecular weight ranging from 25 to 30 kDa for recombinant wild-type human PrP and from 26 to 32 kDa for wild-type bovine PrP. Further, we report here the generation of recombinant mutant prion proteins that are associated with inherited human prion diseases such as fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD). Both mutated variants, the FFI-associated PrP carrying a mutation at amino acid position 178 and the CJD-linked form containing an insertion of nine additional octarepeats reveal proteinase K resistance, one of the typical biochemical properties of the infectious scrapie isoform of the prion protein. By contrast, recombinant wild-type PrP was completely proteinase K sensitive when expressed in SFV-transfected BHK cells. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP. In order to purify recombinant human and bovine PrP from cell lysates, a FLAG-tag was introduced either at the N-terminus behind the signal peptide or at the C-terminus close to the adhesion site of the GPI anchor. N-terminal insertion did not extensively influence the trafficking of the FLAG-tagged protein to the cell surface, whereas insertion close to the GPI attachment site clearly affected the transport of the majority of PrP to the cell membrane, probably resulting in their retention within the secretory pathway. All FLAG-tagged prion proteins were expressed efficiently in BHK cells and showed a typical glycosylation pattern, allowing their rapid and simple purification via anti-FLAG antibody chromatography
Characterization of the 37-kDa/67-kDa laminin receptor as the cell surface receptor for the cellular prion protein
Full text linkPrions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPSc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies (TSEs), which affect both, humans and animals. Human prion diseases occur in infectious, sporadic or genetic forms. The "protein only" hypothesis argues that the key event in the pathogenesis represents the conversion of the normal host protein, PrPc, into its pathogenic isoform PrPSc. Prion diseases have been associated with the accumulation of this abnormally folded protein and its neurotoxic effects. However, it is not known if PrPc loss of function is an important factor since the normal biological function of PrPc, a cell surface-anchored glycoprotein predominantly expressed in neuronal cells, and the cellular processes in which this protein is involved remain obscure.
Recently, the human 37 kDa laminin receptor precursor (LRP), which represents the precursor of the human 67 kDa high-affinity laminin receptor (LR), was identified as a binding partner for the cellular prion protein in a yeast two-hybrid screen. In order to characterize the possible role of LRP/LR as a cell surface receptor for PrPc, cell culture studies were performed to investigate the cellular localization of PrP and LRP/LR and to analyse the binding and internalization behaviour of PrP depending on the presence of LRP/LR on the cell surface of neuronal and non-neuronal cells. Immunofluorescence analysis of non-permeabilized murine neuroblastoma cells demonstrated that PrP and LRP/LR co-localize on the surface of these cells. In addition, baby hamster kidney (BHK) cells transfected with recombinant Semlik-Forest virus RNAs overexpressed human PrP and human LRP at their cell surface, the latter one orientated as a type II transmembrane protein with its C-terminus outside and its N-terminus inside the cell. Co-localization of both proteins was observed on BHK cells co-transfected with LRP and PrP encoding recombinant SFV RNAs. Cell binding and internalization assays with recombinant human PrP demonstrated the LRP/LR-dependent binding and endocytosis of externally added human PrP. An increased, dose-dependent cell binding of recombinant PrP was demonstrated by BHK cells overexpressing full-length human LRP on their cell surface. Trypsin treatment of the cell surface revealed the LRP dependent internalization of GST-tagged and untagged, glycosylated PrP. In contrast to wild-type LRP, the expression of an LRP mutant lacking its transmembrane domain led to the secretion of this mutant from transfected BHK cells and totally abolished the binding and internalization of exogenous, recombinant PrP. This LRP mutant could function as a decoy recetor in therapy of TSEs. The strict LRP/LR specificity of the PrP binding to neuronal cells was verified by testing the displacement capacity of a series of different antibodies in the LRP-PrP binding reaction. Only LRP and PrP specific antibodies were able to block totally the binding of human GST-fused PrP to N2a and NT2 cells whereas various control antibodies used for competition showed no effect. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of cellular PrP to the 37-kDa/67-kDa LRP/LR. The relationship between the 37-kDa LRP and the 67-kDa high-affinity LR is unknown so far. Both forms were observed in plasma membrane fractions of N2a cells. We conclude from these data that the 37-kDa/67-kDa laminin receptor acts as the main cell surface receptor for PrP.
High-level expression and purification of recombinant, glycosylated prion proteins in mammalian cells is essential for a better understanding of the physiological function of PrPc and biochemical processes responsible for prion diseases. Due to the presence of important organelles, membranes and other cellular cofactors which are necessary for the correct processing, trafficking and localization of prion proteins mammalian cell culture systems such as the Semliki-Forest virus (SFV) system allow the synthesis and characterization of wild-type as well as mutant PrP to get a better insight into the biology of these proteins. Therefore, the SFV system was used to generate recombinant highly glycosylated human wild-type and human disease-associated mutant prion proteins as well as FLAG-tagged human and bovine PrP in cultured BHK cells. Both mutated variants, which are related to the human prion diseases fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) reveal proteinase K (PK) resistance, one of the most typical biochemical properties characteristic for the infectious scrapie isoform of the prion protein. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP without any significant differences regarding the cellular distribution and expression level. In addition, FLAG-tagged prion proteins were expressed with high efficiency in BHK cells showing the typical glycosylation pattern allowing the rapid and simple purification via anti-FLAG antibody chromatography.
PrP dimers could play an essential role in the PrPc to PrPSc conversion process and might be involved in PrP interspecies transmission. Recently, crystallization of the prion protein in a dimeric form was reported. Size exclusion chromatography showed that native soluble homogeneous FLAG tagged prion proteins from hamster, man and cattle expressed in the baculovirus system were predominantly dimeric. The PrP/PrP interaction was confirmed in rec. SFV-RNA transfected BHK cells co-expressing FLAG and oligohistidine tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains.
The identification of the 37-kDa/67-kDa laminin receptor as the receptor for the cellular prion protein might represent an important step for a better understanding of the molecular biology of prion diseases and might lead to the development of powerful therapeutics such as LRP/LR specific antibodies for the treatment of these unconventional diseases
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
- …
