1,721,062 research outputs found
Ferritin engineering by chemical modification for bioimaging and drug delivery
Get PDFNegli ultimi anni, le nanoparticelle di ferritina hanno ricevuto crescente attenzione per le loro peculiari proprietà ed il loro impiego per applicazioni diagnostiche e terapeutiche. Le ferritine infatti sono nanoparticelle proteiche stabili, biocompatibili, versatili, uniformi ed omogenee con 24 subunità assemblate in modo da formare una gabbia sferica, con una cavità interna di 8 nm di diametro, in grado di ospitare qualsivoglia molecola. Inoltre le ferritine possono essere internalizzate all’interno delle cellule mediante endocitosi mediata dal recettore TfR1, un recettore largamente sovraespresso nelle cellule cancerose. Per queste caratteristiche le ferritine emergono come nanoparticelle ideali per l’incapsulamento ed il delivery selettivo di varie molecole endogene. Tuttavia le ferritine di mammifero sono assemblate in una forma stabile e chiusa di tetraeicosamero, in grado di dissociare solo in condizioni estreme quali pH acidi potenzialmente dannosi sia per la proteina che per le molecole incapsulate. Al contrario, la ferritina di Archaeaoglobus fulgidus (AfFt) è in grado di dissociare e riassociare dipendentemente dalla forza ionica dell’ambiente in cui si trova. Tale ferritina è stata perciò “umanizzata” mediante tecniche di ingegneria genetica producendo un mutante di superficie di AfFt con un loop esterno in grado di mimare il motivo di riconoscimento della ferritina umana per il recettore TfR1 in cellule umane e con l’architettura e le proprietà di oligomerizzazione tipiche della ferritina di AfFt. Le proprietà di associazione e dissociazione dipendenti dalla forza ionica del sistema sono state attentamente caratterizzate da un punto di vista sia termodinamico che cinetico, tramite funzionalizzazione della proteina con molecole fluorescenti di pirene. Tale fluoroforo è inoltre stato utilizzato per la visualizzazione, mediante la tecnica di microscopia di fluorescenza a due fotoni, dell’uptake di ferritina all’interno di cellule cancerose quali le cellule HeLa. L’effettiva internalizzazione della ferritina dimostra quindi sia l’avvenuto riconosciuto del mutante umanizzato da parte del recettore TfR1 sia il potenziale di tale nanoparticella per applicazioni di microscopia e bioimaging. In aggiunta l’utilizzo della ferritina umanizzata come nano particella per l’incapsulamento ed il delivery selettivo su cellule cancerose di brevi sequenze di DNA è sotto sperimentazione.
In conclusione questi studi dimostrano la potenzialità della ferritina umanizzata come nano particella versatile, facilmente modificabile ed in grado di incapsulare molecole all’interno della cavità in condizioni fisiologiche al fine di trasportare e rilasciare selettivamente la molecola incapsulata all’interno delle cellule tumorali per scopi terapeutici o diagnostici
Supplemental_Material – Supplemental material for OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics
No full textSupplemental material, Supplemental_Material for OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics by Lorena Pochini, Michele Galluccio, Mariafrancesca Scalise, Lara Console and Cesare Indiveri in SLAS Discovery</p
DISC867317_SupplementalMaterial – Supplemental material for SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure
No full textSupplemental material, DISC867317_SupplementalMaterial for SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure by Luca Palazzolo, Chiara Paravicini, Tommaso Laurenzi, Sara Adobati, Simona Saporiti, Uliano Guerrini, Elisabetta Gianazza, Cesare Indiveri, Catriona M. H. Anderson, David T. Thwaites and Ivano Eberini in SLAS Discovery</p
FAD forming and destroying via human FAD synthase: a puzzle of modulated and modulating activities
No full textFAD synthase (FADS or FMN:ATP adenylyl transferase) coded by human FLAD1 gene, is the last enzyme in the pathway converting riboflavin into the redox cofactor FAD.Mutations in FLAD1 gene are responsible for Riboflavin-Responsive and Non-Responsive MADD and Combined Respiratory-Chain Deficiency[1].
Alternative splicing of the FLAD1 gene generates different hFADS isoforms: the mitochondrial isoform 1 and the cytosolic isoform 2. These are bi-functional enzymes containing two domains: a PAPS domain at the C-terminus able to catalyze FAD synthesis (EC 2.7.7.2), a molybdopterin-binding domain at the N-terminus able to perform FAD hydrolysis (EC 3.6.1.18).We show here that theCo2+-dependent hydrolytic activity of hFADS2 is strongly stimulated in the presence of K+, reaching a Vmax even higher than that of FAD synthesis. hFADS2acts as a non-NuDiX hydrolaseand it could interconnect FAD and NAD homeostasis.
Recently,in patients suffering for frameshift mutations in the FLAD1 gene, we revealed ashort transcript variant corresponding to isoform 6,containing the sole PAPS domain[1]. We overproduced and characterized this emergency protein, which is relevant for patient survival. It is able to synthesize, but not to hydrolyze FAD [2].In the aim to find a target for therapy intervention in patients harboring FADS defects, a variant of hFADS6 carrying the site-directed mutation D238A, expected to exhibit a higher Kcat, was also over-produced, purified and characterized
MODULATING CELLULAR FLAVIN COFACTOR LEVELS IN HUMANS VIA THE BI-FUNCTIONAL AND MONOFUNCTIONAL FLAD1 GENE PRODUCTS
No full textFunctional Study of the Human Riboflavin Transporter 2 Using Proteoliposomes System
No full textRiboflavin is essential for cell viability. The biologically active forms of riboflavin, FMN and FAD, participate in many biochemical redox reactions including the metabolism of carbohydrates, amino acids, and lipids. Differently from bacteria, fungi, and plants which synthesize riboflavin, higher organisms have lost the ability to synthesize the vitamin and must absorb it from food and intestinal microflora production. The riboflavin flux through cell membranes occurs via specific transporters belonging to the SLC52 family. Three members of this family have been identified so far which show poor homology with the riboflavin transporters of Saccharomyces cerevisiae or bacteria. Alterations of RFVTs are causative of severe diseases. Indeed, under pathological stress, humans are susceptible of developing riboflavin deficiency. Such a deficiency in pregnancy induces fetus abnormalities, and has been indicated as a risk factor for anemia, cancer, cardiovascular diseases, and neurodegeneration. Moreover, inherited diseases are also of interest; the most well-described is the Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset sensorineural deafness and pontobulbar palsy. Numerous polymorphisms of Slc52a2 and Slc52a3 genes associated with this syndrome have been discovered. In spite of their important metabolic role and their relevance to human health, the riboflavin transporters are still poorly characterized. Bacterial overexpression, purification, and protein reconstitution in liposomes represent an up-to-date methodology for obtaining functional data information. The methodology for reconstituting the RFVT2 into proteoliposomes and performing transport assay is described. These methods will be suitable for investigating the functional defects of the variants of RFVTs associated with human pathologies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Human mitochondrial carnitine acylcarnitine carrier: Molecular target of dietary bioactive polyphenols from sweet cherry (Prunus avium L.)
No full textThe effect of polyphenols, recognized as the principal antioxidant and beneficial molecules introduced with the diet, extracted from sweet cherry (Prunus avium L.) on the recombinant human mitochondrial carnitine/acylcarnitine transporter (CACT) has been studied in proteoliposomes. CACT transport activity, which was strongly impaired after oxidation by atmospheric O2 or H2O2, due to the formation of a disulfide bridge between cysteines 136 and 155, was restored by externally added polyphenols. CACT reduction by polyphenols was time dependent. Spectroscopic analysis of polyphenolic extracts revealed eight most represented compounds in four cultivars. Molecular docking of CACT structural omology model with the most either abundant and arguably bio-available phenolic compound (trans 3-O-feruloyl-quinic acid) of the mix, is in agreement with the experimental data since it results located in the active site close to cysteine 136 at the bottom of the translocation aqueous cavity
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