431 research outputs found
Metallothionein gene family in the sea urchin Paracentrotus lividus: Gene structure, differential expression and phylogenetic analysis
Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization
An intronic cis-regulatory element is crucial for the alpha tubulin Pl-Tuba1a gene activation in the ciliary band and animal pole neurogenic domains during sea urchin development
In sea urchin development, structures derived from neurogenic territory control the swimming and feeding responses of the pluteus as well as the process of metamorphosis. We have previously isolated an alpha tubulin family member of Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the ciliary band and animal pole neurogenic domains of the sea urchin embryo. In order to identify cis-regulatory elements controlling its spatio-temporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs), was identified as responsible for proper gene expression. An enhancer role was ascribed to ICR1 and ICR2, while ICR3 exerted a pivotal role in basal expression, restricting Tuba1a expression to the proper territories of the embryo. Additionally, the mutation of the forkhead box consensus sequence binding site in ICR3 prevented Pl-Tuba1a expression
A modified culture medium for improved isolation of marine vibrios
Marine Vibrio members are of great interest for both ecological and biotechnological research, which often relies on their isolation. Whereas many efforts have been made for the detection of food-borne pathogenic species, much less is known about the performances of standard culture media toward environmental vibrios. We show that the isolation/enumeration of marine vibrios using thiosulfate-citrate-bile salts-sucrose agar (TCBS) as selective medium may be hampered by the variable adaptability of different taxa to the medium, which may result even in isolation failure and/or in substantial total count underestimation. We propose a modified TCBS as isolation medium, adjusted for marine vibrios requirements, which greatly improved their recovery in dilution plate counts, compared with the standard medium. The modified medium offers substantial advantages over TCBS, providing more accurate and likely estimations of the actual presence of vibrios. Modified TCBS allowed the recovery of otherwise undetected vibrios, some of which producing biotechnologically valuable enzymes, thus expanding the isolation power toward potentially new enzyme-producers Vibrio taxa. Moreover, we report a newly designed Vibrio-specific PCR primers pair, targeting a unique rpoD sequence, useful for rapid confirmation of isolates as Vibrio members and subsequent genetic analyses
Carbon nanodots for on demand chemophotothermal therapy combination to elicit necroptosis: Overcoming apoptosis resistance in breast cancer cell lines
Background: Engineered luminescent carbon nanodots (CDs) are appealing nanomaterials for cancer image-guided photothermal therapy combining near infrared (NIR)–triggered hyperthermia, imaging, and drug delivery in a single platform for efficient killing of cancer cells. This approach would allow eliciting synergistic regulated cell death (RCD) routes such as necroptosis, targeting breast cancer cells refractory to apoptosis, thus overcoming drug resistance. Methods: We report the preparation of CDs bearing biotin as a targeting agent (CDs-PEG-BT), which are able to load high amounts of irinotecan (23.7%) to be released in a pulsed on-demand fashion. CDs-PEG-BT have narrow size distribution, stable red luminescence, and high photothermal conversion in the NIR region, allowing imaging of MDA-MB231 and MCF-7 cancer cells and killing them by photothermal and chemotherapeutic insults. Results: Cellular uptake, viability profiles, and RCD gene expression analyses provided insights about the observed biocompatibility of CDs-PEG-BT, indicating that necroptosis can be induced on-demand after the photothermal activation. Besides, photothermal activation of drug-loaded CDs-PEG-BT implies both necroptosis and apoptosis by the TNFα and RIPK1 pathway. Conclusions: The controlled activation of necroptosis and apoptosis by combining phototherapy and on-demand release of irinotecan is the hallmark of efficient anticancer response in refractory breast cancer cell lines in view of precision medicine applications
Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years
Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations
Clinical evolution and quality of life in clinically based copd chronic bronchitic and emphysematous phenotypes: Results from the 1-year follow-up of the storico italian observational study
Introduction: Understanding clinical evolution of chronic obstructive pulmonary disease (COPD) is crucial for improving disease management. Materials and Methods: STORICO (NCT03105999), an Italian, multicenter, non-inter-ventional, observational study conducted in 40 pulmonology centers, aimed to describe the 1-year clinical evolution and health status of clinicallbased phenotypes. Baseline and follow-up data of COPD subjects with a chronic bronchitis (CB) or emphysema (EM) phenotype were collected. The frequency of COPD symptoms during the 24 hours (gathered via the night-time, morning and day-time symptoms of COPD questionnaire) and the anxiety and depression levels (via the HADS Scale) were recorded at each visit. Results: A total of 261 CB and 159 EM patients were analyzed. CB patients with ≥1 night-time symptom seemed to be more frequent (51.7%, 41.8% and 41.4% at baseline, 6-month and 12-month follow-up, respectively) than EM (37.7%, 32.1% and 30.2% at study visits) even if no statistical differences were observed at time points between phenotypes (chi-square test p-values presence/absence of night-time symptoms in CB vs EM at study visits >0.0007). In the first 6 months, the frequency of patients with ≥1 night-time symptom decreased of 9.9% in CB and of 5.6% in EM. A clinically relevant decline of DLCO % predicted over 1 year in EM was observed, the mean (SD) being 61.5 (20.8) % at baseline and 59.1 (17.4) % at 12-month follow-up. EM had higher levels of anxiety and depression than CB (median (25th-75th percentile) HADS total score in CB: 7.0 (4.0–13.0) and 7.0 (3.0–12.0), in EM: 9.0 (3.0–14.0) and 9.5 (3.0–14.0) both at baseline and at 6-month follow-up, respectively), considering 1.17 as minimally clinical important difference (MCID) for the total score. Conclusion: EM patients, evaluated in a real-world setting, seem to suffer from a worse clinical condition and health status compared to CB patients, appearing to have “more treatable” traits
Esperimento pilota mirato all’analisi dell’effetto della stimolazione immunitaria su A. viridis, finalizzato alla produzione di composti biologicamente attivi
L’esperimento mira a valutare l’eventuale attività antibatterica di peptidi prodotti da individui di Anemonia viridis in seguito a stimolazione immunitaria e quindi alla realizzazione di un protocollo riproducibile e ottimizzato per la produzione di tali composti
Applicazione di un protocollo di immuno-stimolazione su individui di Pracambarus clarkii mirato alla produzione di peptidi con attivitĂ biologica
Uno dei principali problemi inerenti l’identificazione e l’analisi di nuovi peptidi naturali di origine marina è la disponibilità in termini quantitativi del materiale bioattivo (Cragg et al., 1997). Infatti, le concentrazioni di tali peptidi negli invertebrati marini sono spesso meno del 10-6 % del peso umido, ed il loro rendimento a seguito delle procedure di estrazione tradizionali risulta ancora più basso. Tuttavia, sono molteplici i fattori che possono influenzare l'esito della identificazione degli Anti Microbial Peptides (AMP), come le variazioni geografiche e stagionali (sito di campionamento), le diverse fasi della vita (specie pelagiche o bentoniche), l'età , il sesso e lo stato fisiologico (malattia, allevamento, muta).
A causa della difficoltà nell’ottenere quantità sufficienti di composti bioattivi, potrebbe essere più produttivo dissezionare in diversi tessuti/organi gli invertebrati selezionati per poi estrarre il materiale da saggiare (Haug et al., 2002a, 2002b, 2004) ciò al fine di separare i peptidi di interesse da migliaia di sostanze inattive. Dividere gli organismi in parti differenti selezionando ciascun tessuto potrebbe anche fornire l'indicazione se l'animale produce da sé il principio attivo o se esso proviene dalla dieta o ancora è associato a parassiti o microrganismi.
La maggior parte degli AMP marini sono stati di fatto isolati dal compartimento sangue, sia dall’emolinfa/fluido celomatico (sangue intero) o dagli emociti/celomociti (cellule del sangue). Alcuni peptidi tuttavia sono stati scoperti e isolati da altri tessuti, ma essendo questi circondati (negli invertebrati marini) dagli emociti/celomociti, è possibile che provengano da questi ultimi.
Inoltre tutte le variabili analitiche hanno una potenziale influenza sul risultato sia in termini quantitativi che qualitativi oltre che per la stabilità del campione anche per la riproducibilità del protocollo. Tra queste variabili ricadono le condizioni di conservazione del campione (tempo trascorso dall’estrazione, la temperatura prima dell’estrazione ed alla separazione, il congelamento e la liofilizzazione), la tipologia del campione ed il metodo di prelievo, la separazione delle cellule dal sangue (velocità di centrifugazione, durata e temperatura), l’uso del tampone (tipo, pH, forza ionica e temperatura), l'uso e il tipo di anticoagulanti e cocktail inibitori della proteasi.
Inoltre, dati bibliografici riportano come la stimolazione immunitaria determini un aumento sia quantitativamente che qualitativamente della produzione di peptidi antimicrobici.
Per tale motivo si è proceduto ad effettuare un esperimento pilota per valutare se in tali condizioni fosse superiore la quantità di piccoli peptidi sintetizzati rispetto ad individui non stimolati e se nei differenti tempi di stimolazione selezionati, emergesse una differenziazione a carico dei peptidi prodotti in relazione ad una possibile attività di natura antibatterica
Lo stato dell’ambiente marino nell’area del Canale di Sicilia in prossimità del SIN di Gela.
Viene illustrata in questa nota l’attuale condizione in cui si trova lo stato dell’ambiente marino nell’area del Canale di Sicilia immediatamente a ridosso della costa merdionale delle Sicilia dal Golfo di Gela alla costa iblea. Lo stato dell’ambiente in oggetto viene valutato in base alla distribuzione di alcuni metalli in tracce normalmente ritenuti fra i più significativi per gli scopi prefissati, esaminati lungo la colonna d’acqua e nei sedimenti superficiali di fondale. I risultati ottenuti, illustrati anche attraverso un’indagine statistica multivariata, indicano un arricchimento superficiale della colonna d’acqua in lantanio che può essere ascritto tanto ad un’origine litogenica, quanto ad input antropico. Nei sedimenti al naturale raggruppamento fra elementi in tracce in tracce basato su analogie comportamentali di carattere geochimica sfugge il Ni che mostra di provenire da una fonte differente per la quale non si può escludere la natura antropica. Tale ipotesi di esiti di antropizzazione trova conforto nei raffronti condotti con il chimismo del particolato atmosferico desunto da dati di letteratura
Expression of adrenomedullin and proadrenomedullin N-terminal 20 peptide in human and rat prostate
Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP)
are two recently discovered hypotensive peptides translated from the same message transcript
(preproAM mRNA). In this article we report the presence of AM, PAMP, and their
mRNA in human and rat prostate and of AM receptor mRNA in rat prostate. PreproAM
mRNA was found in the epithelium of normal human and rat prostate glands by in situ hybridization.
In humans, it was mainly expressed in the basal cells. In rat, its expression was
higher in the ducts than in the acini of all the prostate lobes. Immunocytochemistry identified
a similar distribution pattern for AM compared with its mRNA but showed different locations
for AM and PAMP immunoreactivity. The former was widespread in the epithelia,
whereas the latter was almost exclusively found in neuroendocrine cells. In rat, Western
blot analysis confirmed the presence of high levels of AM peptide in the ventral lobe and
of its precursor in the ventral and dorsolateral lobes. Immunoreactivity for serotonin, chromogranin
A, PAMP, and AM defined four subpopulations of prostate neuroendocrine-like
cells in rat, a cell type that has not been previously described
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