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Trasmissione intraspecie di encefalopatia spongiforme bovina e di encefalopatia spongiforme amiloidotica bovina. Caratterizzazione molecolare e ultrastrutturale di un nuovo ceppo di prione nel bovino
No full textLe Encefalopatie Spongiformi Trasmissibili (TSE) o Malattie da Prioni, costituiscono
un gruppo di malattie neurodegenerative fatali, ad eziologia sporadica, genetica o
infettiva che colpiscono l’uomo e diverse specie animali. Il meccanismo
patogenetico responsabile delle TSE consiste nella conversione conformazionale
della normale proteina prionica cellulare (PrPC), con una struttura
prevalentemente ad α-elica, in una forma aberrante, ricca in β-sheet,
parzialmente insolubile in detergenti non ionici e resistente alla digestione con
proteasi, denominata PrPSc, che rappresenta il marcatore diagnostico di queste
malattie.
Nei bovini, sono due le forme di TSE conosciute: l’encefalopatia spongiforme
bovina (BSE) e l’encefalopatia spongiforme amiloidotica bovina (BASE). Della
prima sono ampiamente note sia la manifestazione fenotipica sia la
caratterizzazione biochimica del ceppo proteico grazie al fiorire di studi in seguito
all’epidemia che a partire dal 1985 ha colpito molti allevamenti bovini della Gran
Bretagna. Della seconda, invece, non esiste ancora una completa
caratterizzazione a causa della sua recente identificazione. Infatti solo nei primi
anni del 2000 sono state riconosciute due nuove forme atipiche di BSE, chiamate
high type (BSE-H) and low type (BSE-L) in base alla differente migrazione
elettroforetica dell’isoforma non glicosilata della PrPSc bovina. Anche la BASE è
stata definita una forma atipica di BSE per le notevoli differenze molecolari e
neuropatologiche presentate dalla proteina prionica. Infatti la PrPSc associata alla
BASE è risultata paragonabile a quella della BSE-L, per il peso e il profilo di
glicosilazione delle isoforme, e distinguibile dalla BSE per la presenza di depositi
ricchi di amiloide, chiamati placche kuru, a livello cerebrale. E’ stata così suggerita
l’ipotesi dell’esistenza di un ceppo di prione della BASE diverso rispetto a quello
della BSE, ma uguale a quello della BSE-L.
Anche recenti studi di trasmissione in topi PrP-bovinizzati hanno contribuito a
confermare tali dati, dimostrando come la BASE sia una patologia più aggressiva e
più facilmente trasmissibile della BSE. Mancando sia le conferme a queste
preliminari sperimentazioni sia l’identificazione del fenotipo clinico-patologico
associato alla BASE, abbiamo progettato uno studio di trasmissione intraspecie di
ceppi di BSE e BASE in bovini di razza Frisona e Bruna Alpina. Utilizzando la stessa
specie di quella che ha contratto in origine l’infezione, si sono raggiunti due
obiettivi: ridurre il periodo di incubazione, evitando il fenomeno dell’adattamento
e tutti i processi atti a superare la barriera di specie, e delineare il quadro
fenotipico della BASE. Gli animali inoculati con BASE hanno manifestato un
comportamento affine alla depressione diverso da quello più irrequieto degli
animali infettati con BSE, associato ad una progressiva amiotrofia. Inoltre i bovini
hanno presentato profili molecolari e neuropatologici affini alle caratteristiche del
donatore, confermando la diversità dei due strains proteici. Lo studio si è avvalso
anche di un’analisi ultrastrutturale, che, oltre a confermare la diversità del pattern
di deposizione della PrPSc nelle due forme di TSE, ha rilevato significative
differenze nella localizzazione subcellulare della proteina prionica.
Questa sperimentazione ha permesso di valutare tutti gli aspetti clinici, biochimici,
istologici, immunoistochimici e ultrastrutturali associati alla PrPSc ed ottenere
quindi una completa caratterizzazione della BASE, confermando i dati già presenti
in letteratura sulla diversità del ceppo proteico rispetto quello della BSE e
riproducendo per la prima volta la manifestazione fenotipica.Transmissible Spongiform Encephalopathies (TSE) or Prion diseases are a group
of rare, fatal and transmissible neurodegenerative disorders that affect both
humans and animals.
Clinically these diseases exist in sporadic, genetic and acquired forms, and
present with a variety of neurological signs. TSE diseases are characterized by
accumulation, primarily in the brain, of an abnormal isoform of the normal
host-encoded prion protein (PrPC), named PrPSc that is considered the
disease-associated agent. The central event of the TSE is a post-translational
conformational change of the PrPC, a plasma membrane glycoprotein, rich in
α-helix, into the PrPSc that has a higher β-sheet content.
In bovine there are two forms of TSE: Bovine Spongiform Encephalopathy (BSE)
and Bovine Amyloidotic Spongiform Encephalopathy (BASE). After the epidemy in
cattle in UK in 1985, many researches characterized the phenotype and the
biochemistry of PrPSc in BSE. Recently new atypical forms of BSE are discovered
and called BSE-H (higher) and BSE-L (lower) for the different molecular mass of
unglycosilated isoform of PrPSc. Also BASE is classified as atypical BSE with a prion
protein similar to BSE-L and it is distinguishable from BSE for differences in
pattern of deposition, consistent in the presence of amyloid plaques, in distinct
molecular masses and in glycosilation profile of prion isoforms. These
observations suggest that cattle may have two distinct prion strains, refered to
two forms of TSE and that prion protein of BASE could be the same of BSE-L.
Also recent studies on PrP-bovinized transgenic mice have showed that BASE is a
more aggressive disease than BSE, characterized by shorter period of incubation.
To investigate the characteristics of prion strains responsible of BSE and BASE, we
carried out transmission by inoculation of BSE and BASE isolates in Fresian and
Alpine brown cattle. Intraspecies transmission permitted firstly to reduce the
period of incubation, overcoming the species barrier, and secondly to identify the
phenotype of BASE. BASE inoculated cattle showed a progressive muscle atrophy
and a dull behaviour, in contrast with aggressiveness and hypersensitivity of
BSE-infected animals. The prion strains of inoculi preserved their different
biochemical and neuropathological properties also after transmission. Finally, a
more detailed ultrastructural analysis confirmed the distinction in patterns of PrPSc
deposition and it showed different subcellular localization of prion protein
between BSE and BASE infected bovine.
By evaluation of biochemical, immunoistochemical and ultrastructural results, our
study gave an improvement in the characterization of BASE prion strain,
describing the clinical phenotype
The peel and pulp of mango fruit: A proteomic samba
No full textCombinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of mango peel and pulp as well their peptidome content (the latter as captured with a C18 resin). The aim of this study was not only to perform the deepest investigation so far of the mango proteome, but also to assess the potential presence of allergens and of peptides endowed with biol. activities. The proteins of peel and pulp have been captured under both native and denaturing extn. techniques. A total of 334 unique protein species have been identified in the peel vs. 2855 in the pulp, via capture with CPLLs at different pH values (2.2 and 7.2
Proteomic fingerprinting of protein corona formed on PEGylated multi-walled carbon nanotubes
Full text linkIn Nanomedicine, carbon-based nanomaterials, like Carbon Nanotubes (CNT), are considered potential candidates
as drug delivery systems. In vivo adsorption of physiological proteins onto carbon nanotubes, through
noncovalent interactions, forms a protein corona or bio corona, able to influence biological properties and
biocompatibility of CNT. This study aimed to explore the composition of protein corona formed onto PEGylated
Multi-Walled Carbon Nanotubes (MWCNT-PEG5k), after their incubation in human plasma. Plasma proteins
were sequentially eluted in different conditions by using both native and denaturant buffers, useful to characterize
soft and hard corona. Proteomic methods and mass spectrometry analysis have identified proteins in soft
corona, involved in the regulation of immune response and in the CNT transport, and biomolecules in hard corona
with a role in the maintenance of host homeostasis. These promising results have demonstrated the potential of
PEGylated Multi-Walled Carbon Nanotubes as future candidates for drug delivery
A Stepwise Approach for the Isolation and the Identification of Chemically Reactive Exofacial Protein Thiols
No full textThe plasma membrane controls the selective internalization of (macro)molecules through different mechanisms, often relaying on specialized outward-facing carriers such as exofacial proteins thiols (EPTs). Although the interchange of critical thiols and disulphides between EPTs and exogenous cargoes is the first critical event, the identification of specific cell interactors remains to be thoroughly explored. Besides, it is likewise evident that only the relatively little suite of EPTs truly reactive can be considered theranostic targets
Plucking, pillaging and plundering proteomes with combinatorial peptide ligand libraries
No full textRecent developments in the technique of combinatorial peptide ligand libraries, for enhancing the visibility of the low-abundance proteome, are reviewed here. Novel en bloc elution systems, allowing essentially complete proteome recovery in a single step, are reported here, particularly, en bloc elution with 3–5% boiling sodium dodecyl sulphate (SDS) or in urea–thiourea–CHAPS added with either 40 mM formic acid or 25 mM cysteic acid. Novel capturing systems are also discussed: in particular, although capturing at pH 7.2 in physiological saline has always been recommended, it is shown that capturing also at acidic (pH 3.8) and alkaline (pH 9.5) values substantially increments the total captured protein population. Some examples of detection of novel proteins by the described methodology are also discussed. In particular, in the case of venom proteins, where essentially all components had been detected and fully described by conventional means, the application of the ligand library technology allowed the discovery of two, previously unreported, trace enzymes necessary for the maintenance of the native structure of venom components, namely peroxiredoxin and glutaminyl cyclase. In the case of the red blood cell (RBC) cytoplasmic proteome, where a grand total of 1570 components of the 2% minority proteomes have been identified, these findings allowed to unravel the genetic defect of a rare RBC disease, called congenital dyserythropoietic anemia type II. The mutations are located in the SEC23B gene coding for the SEC23B protein, detected for the first time in the RBC proteome thanks to the peptide capturing technolog
Proteomic investigation on bio-corona of functionalized multiwalled carbon nanotubes
No full textBackground: The formation of bio-corona, due to adsorption of biomolecules onto carbon nanotubes (CNTs) surface in a physiological environment, may lead to a modified biological “identity” of CNTs, contributing to determination of their biocompatibility and toxicity. Methods: Multi-walled carbon nanotubes surfaces (f-MWCNTs) were modified attaching acid and basic chemical functions such as carboxyl (MWCNTs-COOH) and ammonium (MWCNTs-N) groups respectively. The investigation of interactions between f-MWCNTs and proteins present in biological fluids, like human plasma, was performed by electrophoretic separation (SDS-PAGE) and mass spectrometry analysis (nLC-MS/MS). Results: A total of 52 validated proteins was identified after incubation of f-MWCNTs in human plasma. 86% of them was present in bio-coronas formed on the surface of all f-MWCNTs and 29% has specifically interacted with only one type of f-MWCNTs. Conclusions: The evaluation of proteins primary structures, present in all bio-coronas, did not highlight any correlation between the chemical functionalization on MWCNTs and the content of acid, basic and hydrophobic amino acids. Despite this, many proteins of bio-corona, formed on all f-MWCNTs, were involved in the inhibitor activity of serine- or cysteine- endopeptidases, a molecular function completely unrevealed in the human plasma as control. Finally, the interaction with immune system's proteins and apolipoproteins has suggested a possible biocompatibility and a favored bio-distribution of tested f-MWCNTs. General significance: Considering the great potential of CNTs in the nanomedicine, a specific chemical functionalization onto MWCNTs surface could control the protein corona formation and the biocompatibility of nanomaterials
Capturing and Amplifying Impurities from Recombinant Therapeutic Proteins Via Combinatorial Peptide Libraries: A Proteomic Approach
No full textA review. The technique of combinatorial peptide ligand libraries (CPLL), for capturing and amplifying low-abundance proteins in r-DNA products as well as in a no. of other biol. systems, is here analyzed in depth and reviewed. This methodol. is based on a creation of several millions of bio-specific ligands composed of hexapeptides produced in a combinatorial way. When acting on an overloading and satn. principle, high-abundance species are captured in limited amts., whereas low-abundance ones keep being concd. on their bio-specific ligand till substantial harvesting from soln. (the capture process occurring in general from ca. 50% up to 90% efficiency). Examples are given on tracking host-cell impurities present in, e.g., recombinant albumin or monoclonal antibodies. Addnl., other examples of detecting traces of additives and fining agents in such beverages as white and red wines are presented. The unique mechanisms underlying the protein capture in the CPLL methodol., as opposed to capture by homogeneous beads, as represented by ion-exchangers and by hydrophobic resins, are discussed in depth
Combinatorial peptide ligand libraries: The conquest of the 'hidden proteome' advances at great strides
No full textA review. The combinatorial peptide ligand library (CPLL) is compared here with the immuno-depletion method for evaluating their resp. abilities in digging deeper and deeper into the low-abundance proteome. A recent report suggested in fact that immuno-subtraction for biomarkers discovery in sera does not perform so well, since it results in a meagre 25% increase in identified proteins compared with unfractionated plasma, leaving little capacity to sample lower abundance proteins. On the contrary, CPLLs permit from 300 to 600% increments in detection abilities, as amply demonstrated in several reports. Moreover, when dealing with large sample vols., an amplification factor of up to four orders of magnitude for trace proteins could be demonstrated, with 80% capture efficiencies even in large (up to 1 L) sample vols. At present, the lower detection ability of CPLLs has been evaluated at 1 ng/mL (traces of casein additives in white wines
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