1,720,969 research outputs found
Analisi funzionale di isoforme native e mutanti della pompa Ca2+-ATPasi della membrana plasmatica
Full text linkThe plasma membrane Ca2+-ATPase represents a primary system for the extrusion of Ca2+ ions from all eukaryotic cells. The PMCA pumps are the product of a multigenic family: 4 genes encode 4 different isoforms (PMCA1-4) and this diversity is further increased by mechanism of alternative splicing of the primary transcripts generating more than 30 variants. The level expression of splice variants changes during development and cell differentiation. PMCA1 and 4 are expressed ubiquitously while PMCA2 and 3 are mostly expressed in the central nervous system although they are also found in the skeletal muscle. The functional meaning of the existence of such a high number of isoforms of PMCA pumps has to be explored. General opinion is that each isoform plays a specific role depending on the specific needs of the cell. Furthermore it has been speculated that the specific cell or tissue localisation of the different isoforms is related to isoform specific interactors: specific partners could regulate the specific distribution of the isoforms and their activity as well.
Previous work in our Laboratory has demonstrated that one of the ubiquitous isoform, PMCA4, in contrast to the tissue-specific isoform PMCA2 interacts with a particularly interesting partner, the 14-3-3 protein, which has an inhibitory effect on the activity of the pump.
In the first part of my PhD program, the study of molecular partners was extended to the remaining isoforms, the tissue-specific PMCA3 pump and the ubiquitous PMCA1 pump. The research was performed through an interaction assay with the yeast two hybrid method using as a “bait” the N-terminal portion of PMCA3 and PMCA1. We found that the other tissue-specific isoform, PMCA3, by contrast with PMCA2, interacts with the 14-3-3 protein, while PMCA1 did not. Our research was than focused on identifying the reason why PMCA2 is the only isoform among the PMCA pumps that does not interact with 14-3-3 protein. Bioinformatics analysis of the N-terminal region of the 4 isoforms of PMCA, used as a bait in the yeast two hybrid assay, has revealed that all isoforms contain a consensus sequence for the binding of 14-3-3 proteins, but also that in the flanking region of this site the sequence of the PMCA2 pump contains two particular aminoacids that disturb the formation of the secondary structure necessary for the interaction. The hypothesis proposed by the bioinformatic analysis was confirmed experimentally. We constructed 2 baits for the yeast two hybrid assay by splitting in two equal portions the original bait of the N-terminal 90 aminoacids of the PMCA4 pumps that interacts with 14-3-3. One of the two probes contained the consensus site for the interaction, but not the sites flanking the consensus site which are responsible for the formation of the secondary structure of ?-helix that, on the basis of the bioinformatics analysis, has been proposed necessary for the interaction.. The other bait did not contain the consensus site for the interaction but contained the flanking region. The ability of the two baits to interact with the 14-3-3 protein was tested in a two hybrid system and none of them gave positive results. A third bait, including the consensus site and the flanking region but shorter than the original 90 aa bait, was also tested and it gave positive results confirming that both the portions included are necessary for the interaction and that the fact that the two short baits fail to interact is not dependent by their reduced length.
The next studies during my PhD program were concentrated to functionally characterize the PMCA2 pumps activity. This isoform is particularly interesting for different reasons: it has peculiar properties that distinguish it from other isoforms. It is also the only pump for which single point-mutations have been reported to generate a pathological phenotype: mice harbouring spontaneous mutations of the gene that encodes for PMCA2 display a phenotype associated to hearing loss and defects in coordination and balance.
The data obtained on the functional characterization of different splice variants of PMCA2 pump are presented in the second part of my thesis. The pumps were overexpressed in the natural environment of model cells and their ability to counteract the transient increase of Ca2+ concentration induced by a physiological stimulus was measured. The activity of some PMCA2 mutants identified in mice and humans has been also characterized.
Mammalian expression plasmids for splice variants w/a, w/b, z/a e z/b of PMCA2 of PMCA2 pump were co-transfected with a plasmid for the expression of a Ca2+ sensitive probe, the recombinant photoprotein aequorin (cytAEQ), in a stable cell line of hamster ovary (CHO). CHO cells were stimulated with ATP, a inositol, 1,4,5 triphosphate (InsP3)-linked agonist that acts on P2Y purinergic receptors coupled to G proteins and generates the production of the second messenger InsP3 (which opens the Ca2+ channels localized in the membranes of the Ca2+ intracellular stores), and a consequent transient increase of the cytosolic Ca2+ concentration. It was observed that isoforms z/b, z/a e w/b were particularly effective in reducing the height of the peak of the Ca2+ transient (they reduce it by 50%), reflecting the capacity of these splicing variants to respond with a rapid activation to the sudden increase of cytosolic Ca2+ concentration. By contrast, the doubly spliced variant w/a has a reduced ability to control the peak of Ca2+: it only reduces it by 30% compared to control cells. The PMCA2w/a is less able to respond efficiently to a sudden increase of cytosolic Ca2+ concentration: this characteristic could justify its exclusive presence in the stereocilia of the hair cells of the inner ear. Indeed, the concentration of extracellular Ca2+ in the endolymph, the liquid that bathes the stereocilia, is significantly lower than those of the other extracellular fluids being in the order of 10-20μM instead of mM.
The characterization of the mutated pumps responsible for the phenotype of deafness in mice (3 mutants) and humans (1 mutants) has shown that the mutations do not affect the ability of the pump to counteract the height of the Ca2+ peak generated by cell stimulation, but rather reduce the activity of the pump in restoring Ca2+ basal levels: the mutations affect the declining phase of the Ca2+ transient. Thus, the basal levels of Ca2+ concentrations are restored more slowly, and, as a consequence, the cells that have the mutated PMCA2 pump are exposed to high cytosolic Ca2+ concentration longer than control cells. The data show that the PMCA2 mutations do not affect the capacity of the pump to respond to a sudden arrival of Ca2+, but rather affect the basal activity of the pump. These alterations of the Ca2+ homeostasis have probably deleterious consequences on the phenomenon of adaptation of the sensory cells of the inner ear, that become “poorly ready” to receive repetitive sound stimuli and thus generate deafness.Le pompe PMCA (Plasma Membrane Ca2+-ATPases) rappresentano un sistema di importanza primaria per l’estrusione del Ca2+ dal citoplasma delle cellule eucarioti.
Le pompe PMCA fanno parte di una famiglia multigenica: 4 geni codificano 4 diverse isoforme (PMCA1-4) e la diversità delle isoforme è aumentata da meccanismi di splicing alternativo dei trascritti primari che generano più di 30 isoforme diverse. L’espressione delle diverse isoforme, oltre ad essere tessuto specifica, è regolata durante lo sviluppo ed il differenziamento cellulare. Le isoforme PMCA1 e 4 hanno una distribuzione ubiquitaria, mentre le isoforme PMCA2 e 3 sono prevalentemente neuronali .
Il significato funzionale di un numero così elevato di isoforme è tuttora oggetto di studio nel campo delle pompe PMCA. Opinione generale è che ognuna delle isoforme svolga un ruolo specifico a seconda delle esigenze specifiche della cellula. Si ipotizza inoltre che la localizzazione e l’attività tessuto-specifica delle diverse isoforme possa essere influenzata da interazioni isoforma-specifiche con partner proteici diversi. I risultati ottenuti nel nostro Laboratorio hanno dimostrato che una delle isoforme ubiquitarie, la pompa PMCA4, a differenza dell’isoforma tessuto-specifica PMCA2, interagisce con un partner particolarmente interessante, la proteina 14-3-3, e che questa interazione ha un effetto inibitorio sull’attività della pompa. Nella prima parte del mio Dottorato di ricerca, l’indagine della ricerca di interattori molecolari è stata estesa alle rimanenti isoforme, quella tessuto-specifica PMCA3 e quella ubiquitaria PMCA1. La ricerca è stata condotta mediante un saggio di interazione di doppio ibrido in lievito usando come “esca” la porzione N-terminale delle pompe PMCA3 e PMCA1. E’ stato riscontrato che l’altra isoforma tessuto-specifica, la pompa PMCA3, a differenza della pompa PMCA2 interagisce con la proteina 14-3-3. La pompa PMCA1 invece non interagisce. La nostra ricerca si è quindi focalizzata nell’individuare il motivo per cui la PMCA2 è l’unica isoforma tra le PMCA a non interagire con la proteina 14-3-3. Un’analisi bioinformatica della regione N-terminale delle 4 isoforme delle PMCA usata come esca nel saggio del doppio ibrido in lievito ha rivelato che in tutte e 4 le isoforme si trova una sequenza consenso per il legame delle proteine 14-3-3, ma anche che la pompa PMCA2 possiede dei residui amminoacidi nelle regioni fiancheggianti il sito di consenso che disturbano la corretta struttura secondaria necessaria alla interazione. L’ipotesi proposta dall’analisi bionformatica è stata confermata sperimentalmente. Sono state infatti costruite 2 esche per il saggio di interazione del doppio ibrido dividendo in 2 la sonda originale di 90aa nella parte N-terminale della PMCA4 che interagiva con 14-3-3. Una delle due sonde conteneva il sito consenso di interazione, ma non i siti di fiancheggianti responsabili della formazione della struttura secondaria ad ?-elica,, l’altra non conteneva il sito di interazione. Queste sonde sono state testate per loro capacità di interagire con la proteina 14-3-3 nel sistema del doppio ibrido in lievito e nessuna delle due ha dato risultati positivi. Una terza sonda, più corta della esca originale di 90 aa ma che conteneva sia il sito di consenso che le regioni fiancheggianti ha dato invece risultati positivi, confermando così che sia il sito di consenso che le regioni fiancheggianti sono necessarie all’interazione e che la non interazione non dipende dalla lunghezza ridotta delle sonde.
Gli studi successivi nel corso del mio dottorato di ricerca si sono quindi concentrati a caratterizzare funzionalmente la pompa PMCA2. Questa isoforma è particolarmente interessante in quanto possiede alcune proprietà che la distinguono dalle altre isoforme. E’ inoltre l’unica isoforma per la quale sono state descritte mutazioni puntiformi del gene responsabili di un fenotipo patologico: topi che presentano mutazioni spontanee del gene della PMCA2 presentano un fenotipo associato a sordità e a difetti di equilibrio e coordinazione. Nella seconda parte della tesi sono presentati i dati ottenuti della caratterizzazione funzionale delle diverse varianti di splicing della pompa PMCA2. Le pompe sono state sovraespresse in un sistema cellulare omogeneo ed è stata misurata la loro capacità di contrastare l’aumento transiente della concentrazione di Ca2+ citosolico indotto da uno stimolo fisiologico. Successivamente è anche stata caratterizzata l’attività di alcune forme mutanti della PMCA2 individuate nel topo e nell’uomo.
I plasmidi di espressione per le varianti di splicing della PMCA2 w/a, w/b, z/a e z/b sono stati co-transfettati con un plasmide per l’espressione di una sonda per il Ca2+, la fotoproteina ricombinante Ca2+-sensible equorina (cytAEQ), in una linea stabile di cellule di ovario di criceto (CHO). Le cellule CHO sono state stimolate con ATP, un agonista fisiologico che agisce sui recettori purinergici P2Y accoppiati a proteine G e genera, in seguito alla produzione del secondo messaggero inositolo 1,4,5 trifosfato (che apre i canali per il Ca2+ dei depositi intracellulari) un aumento transiente della concentrazione di Ca2+ citosolico. E’ stato osservato che le isoforme z/b, z/a e w/b sono particolarmente efficaci nel ridurre l’altezza del picco del transiente di Ca2+ (lo riducono infatti del 50%), caratteristica che riflette la capacità della pompa di rispondere con una rapida attivazione all’aumento improvviso della concentrazione di Ca2+. La variante w/a invece sembra avere una minore capacità di controllare il picco di Ca2+, lo riduce infatti solo del 30% rispetto alle cellule di controllo. La PMCA2w/a quindi risponde meno efficientemente ad un aumento improvviso della concentrazione di Ca2+ citoplasmatica. Questa caratteristica potrebbe giustificare l’esclusiva presenza della variante w/a della PMCA2 nelle stereocilia delle cellule sensoriali dell’orecchio interno. Infatti la concentrazione di Ca2+ extracellulare nell’endolinfa, il liquido che bagna le stereocilia, è notevolmente più bassa di quella degli altri ambienti extracellulari essendo dell’ordine di 10-20 ?M anziche mM.
La caratterizzazione delle pompe mutate responsabili di fenotipo di sordità nel topo (3 mutanti) e nell’uomo (un mutante) ha dimostrato che le mutazioni non modificano le capacità della pompa di contrastare l’altezza del picco del transiente di Ca2+ generato dalla stimolazione cellulare, ma riducono invece la sua velocità nel ripristinare i livelli basali: colpiscono cioè la fase discendente del transiente di Ca2+. I livelli basali di Ca2+ vengono quindi ripristinati più lentamente e ciò determina che nelle cellule che presentano le PMCA2 mutate la concentrazione di Ca2+ citosolica resta elevata per tempi più lunghi rispetto alla situazione che si verifica nelle cellule di controllo. I dati dimostrano quindi che le mutazioni della PMCA2 non influiscono sulla capacità della pompa a rispondere ad un arrivo repentino di Ca2+, ma invece alterano l’attività basale della pompa. Queste alterazioni dell’omeostasi del Ca2+ a livello delle cellule sensoriali dell’orecchio interno si ripercuotono probabilmente sul fenomeno dell’adattamento rendendo le cellule “meno pronte” a ricevere stimoli sonori successivi portando così ad un fenotipo di sordità
Neurofilament light chain as a biological marker for amyotrophic lateral sclerosis: a meta-analysis study
No full textAim: The aim of the present metanalysis is to evaluate blood and CSF Neurofilament light chain (NfL) concentrations in ALS patients, compared to healthy controls, ALS mimic disorders (ALSmd) and other neurological diseases (OND), and to evaluate their diagnostic yield against ALSmd. Methods: Search engines were systematically investigated for relevant studies. A random effect model was applied to estimate the pooled standard mean difference in NfL levels between ALS and controls and a bivariate mixed-effects model was applied to estimate their diagnostic accuracy on blood and CSF. Results and conclusions: NfL CSF levels were higher in ALS compared with all other control groups. On blood, NfL levels were significantly higher in ALS patients compared with healthy controls and ALSmd. In a subgroup analysis, the use of SIMOA yielded to a better differentiation between ALS and controls on blood, compared with ELISA. Studies performed on CSF (AUC = 0.90) yielded to better diagnostic performances compared with those conducted on blood (AUC = 0.78). Further prospective investigations are needed to determine a diagnostic cutoff, exploitable in clinical practice
Plasma-membrane calcium pumps and hereditary deafness
No full textIn mammals, four different genes encode four PMCA (plasma-membrane Ca(2+)-ATPase) isoforms. PMCA1 and 4 are expressed ubiquitously, and PMCA2 and 3 are expressed predominantly in the central nervous system. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of so many isoforms is not clear, but evidently it must be related to the cell-specific demands of Ca(2+) homoeostasis. Recent studies suggest that the alternatively spliced regions in PMCA are responsible for specific targeting to plasma membrane domains, and proteins that bind specifically to the pumps could contribute to further regulation of Ca(2+) control. In addition, the combination of proteins obtained by alternative splicing occurring at two different sites could be responsible for different functional characteristics of the pumps
Unveiling the SOD1-mediated ALS phenotype: insights from a comprehensive meta-analysis
No full textBackground and objectives Amyotrophic lateral sclerosis associated with mutations in SOD1 (SOD1-ALS) might be susceptible to specific treatment. The aim of the study is to outline the clinical features of SOD1-ALS patients by comparing them to patients without ALS major gene variants and patients with variants in other major ALS genes. Defining SOD1-ALS phenotype may assist clinicians in identifying patients who should be prioritized for genetic testing.Methods We performed an extensive literature research including original studies which reported the clinical features of SOD1-ALS and at least one of the following patient groups: C9ORF72 hexanucleotide repeat expansion (C9-ALS), TARDBP (TARDBP-ALS), FUS (FUS-ALS) or patients without a positive test for a major-ALS gene (N-ALS). A random effects meta-analytic model was applied to clinical data extracted encompassing sex, site and age of onset. To reconstruct individual patient survival data, the published Kaplan-Meier curves were digitized. Data were measured as odds ratio (OR) or standardized mean difference (SMD) as appropriate. Median survival was compared between groups.Results Twenty studies met the inclusion criteria. We identified 721 SOD1-ALS, 470 C9-ALS, 183 TARDBP-ALS, 113 FUS-ALS and 2824 N-ALS. SOD1-ALS showed a higher rate of spinal onset compared with N-ALS and C9-ALS (OR = 4.85, 95% CI = 3.04-7.76; OR = 10.47, 95% CI = 4.32-27.87) and an earlier onset compared with N-ALS (SMD = - 0.45, 95% CI = - 0.72 to - 0.18). SOD1-ALS had a similar survival compared with N-ALS (p = 0.14), a longer survival compared with C9-ALS (p < 0.01) and FUS-ALS (p = 0.019) and a shorter survival compared with TARDBP-ALS (p < 0.01).Discussion This study indicates the presence of a specific SOD1-ALS phenotype. Insights in SOD1-ALS clinical features are important in genetic counseling, disease prognosis and support patients' stratification in clinical trials
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
- …
