549 research outputs found
Data on four apoptosis-related genes in the colonial tunicate Botryllus schlosseri
No full textThe datadescribed are related to the article entitled “Recurrent phagocytosis-induced apoptosis in the cyclical generatio nchange of the compound ascidian Botryllus schlosseri” (Franchi et al.,2016) [1]. Four apoptosis-related genes, showing high similarity with mammalian Bax(a member of the Bcl-2 protein family), AIF1 (apoptosis-inducing factor-1), PARP1 (poly ADP ribose polymerase-1) and IAP7 (inhibitor of apoptosis-7) were identified from the analysis of the trascriptome of B. schlosseri. They were named BsBax, BsAIF1, BsPARP1 and BsIAP7. Here, their deduced amino acid sequence were compared wit hknown sequences of orthologous genes from other deuterostome species together with a study of their identity/similarity
Individuazione e caratterizzazione di geni per metallotioneine e altre proteine detossificanti in ascidie
No full textIn order to study the evolution of metallothioneins (MTs) in deuterostomes I have analysed these proteins in Tunicates, which are invertebrate chordates, sister-group of vertebrates. I have isolated the complete cDNA sequences of Ciona intestinalis, Molgula manhattensis and Ascidiella aspersa MTs. Their deduced amino acid sequences are similar in length (39 to 41 amino acids) and each protein has about 30% of cysteine residues which are organised in the typical clusters of vertebrate MTs.
Afterword, my investigation was mainly focused on C. intestinalis, a model tunicate of which the whole genome is available. Nevertheless, no MTs had been previously annotated. After the identification of MT, the research has followed two pathways. The first one was aimed at identifying the tissues involved in the transcription and the gene expression profiles in response to heavy metals such as Zn, Cd and Cu. As regards this last point, in addition to MT, I also considered genes for phytochelatin synthase (PCS), Cu/Zn superoxide dismutase (Cu/Zn SOD), glutamate-cysteine ligase (GCLC) catalytic subunit, glutathione synthetase (GS), glutathione peroxidase 7 (GPX7) and proliferating cell nuclear antigen (PCNA), looking for relationships between them. In order to define their possible evolution, the second approach involved bioinformatic analyses, aimed at understanding the organisation of the gene structure, promoter regions and sequence identity of MTs and of all the other considered proteins.
The deduced protein sequence of C. intestinalis MT (CiMT-1) is quite different from the other deuterostome MTs: it is the shortest MT so far identified in this group, with only 39 amino acids in comparison with 61-68 amino acids of the other taxa. In addition, the KKS motif, that links the two domains (α and β) of the vertebrate MTs, is completely missing. The identity between CiMT-1 and other deuterostome MTs is quite low, ranging from 18.8 and 38.3%.
Vertebrate MT genes show a tripartite structure, with three exons and two introns. Introns differ in length, but their relative position is conserved: the first intron interrupts the sequence at amino acid 9, while the second intron at amino acid 31 or 32, at the junction of the α and β domains. CIMT-1 shows the same tripartite structure, with the first intron in the typical vertebrate position, while the second intron is located in the 3’UTR, as in echinoderm MTs.
The promoter region of CIMT-1 shows features present in both vertebrates and in echinoderms: it contains three half-AREs (antioxidant responsive element) as in echinoderms, and one typical vertebrate ARE. Four MREs (metal responsive elements) are also present.
Cd exposure increases the transcription of CIMT-1 in a non linear way: an initial increase, at 6 h, is followed by a decrease to the control levels at 24 h, then another increase is present after 96 h. Treatments with Zn and Cu, however, induce the gene transcription progressively up to the end of treatment (120 h) when the messenger levels are fourfold the control values.
CiMT-1 mRNA is present only in haemocytes, specifically in granulocytes. These cells, after 24 h of treatment with Cd, are present in tunic and their amount is twice that of controls. Therefore, the decrease of transcript at 24 h may be a result of this cell migration. The subsequent increase is presumably due to specific cellular proliferation of granulocytes in the blood lacunae. This scenario is confirmed by the high rate of cell death in tunic, where haemocytes accumulate, and also by the massive transcription of PCNA in treated samples. Indeed, both CiMT-1 and PCNA show a peak of expression at 96 h. The results obtained with phytochelatin synthase fit with this hypothesis.
In C. intestinalis, PCS is expressed in granulocytes and the mRNA levels remain comparable to those of controls up to 72 h, but then they rise up to 96 h. From literature data it is known that this gene is not inducible, but it is activated in presence of divalent metal ions. Again, it is very likely that the observed messenger peak is not a consequence of an induction phenomenon, but of a strong proliferation of granulocytes.
This is the first data on the transcription of this gene, on the location at granulocyte level and on its involvement in metal detoxification processes in deuterostomes.
The study of the behaviour of the genes involved in detoxification suggests that the enzymes (SOD, GPX) may have a marginal role, whereas the cystein-rich molecules (PCS, GS, GCL) play an important role in presence of Cd, Cu and Zn. Moreover, the localisation of these transcripts, by in situ hybridisation, suggests that circulating haemocytes are the cells mostly involved in detoxification processes.Lo scopo di questa ricerca è quello di analizzare le metallotioneine (MT) nei Tunicati, taxon il sister-group dei Vertebrati, allo scopo di studiare l’evoluzione delle metallotioneine nei deuterostomi invertebrati. Ho quindi isolato sequenze complete di cDNA per MT nei Tunicati Ciona intestinalis, Molgula manhattensis e Ascidiella aspersa. In questi organismi le MT presentano sequenze aminoacidiche dedotte simili, di lunghezza compresa tra 39 e 41 aminoacidi e con circa il 30% di residui cisteinici organizzati nei tipici cluster conservati delle MT dei Vertebrati.
La mia indagine si è quindi concentrata su C. intestinalis, organismo modello dei Tunicati per il quale è disponibile la sequenza dell’intero genoma. Nonostante questo, nessuna MT era stata annotata. Dopo aver identificato la MT, la ricerca si è orientata su due percorsi. Il primo percorso era volto a comprendere quali fossero i tessuti coinvolti nella trascrizione di questo gene e quali ne fossero i profili di espressione in risposta a metalli pesanti quali Zn, Cd e Cu. In quest’ambito ho anche considerato la fitochelatino sitetasi (PCS), la Cu/Zn superossido dismutasi (Cu-Zn SOD), la glutammato cistein-ligasi (GCLC) subunità catalitica, la glutatione sintetasi (GS), la glutatione perossidasi 7 (GPX7) e l’antigene nucleare di proliferazione cellulare (PCNA), al fine di valutarne le relazioni. Il secondo approccio, di tipo bioinformatico, era volto a chiarire l’organizzazione genica, la struttura delle regioni promotrici e le identità di sequenza delle metallotioneine e delle altre proteine studiate, al fine di far luce sulla possibile evoluzione delle sequenze prese in esame.
Le sequenza proteica dedotta da C. intestinalis (CiMT-1) è decisamente diversa dalle MT degli altri deuterostomi: essa risulta essere la più corta MT fino ad ora individuata in questo gruppo, presentando 39 aminoacidi contro 61-68 degli altri taxa. Inoltre, manca completamente il motivo KKS che connette i due domini (alpha e beta) delle MT dei vertebrati. Si evidenziano quindi bassi livelli di identità aminoacidica, tra 18,8% e 38,3%, con le MT degli altri deuterostomi.
Tutte le MT dei vertebrati hanno struttura genica tripartita, con tre esoni e due introni. Gli introni differiscono in lunghezza, ma la loro posizione relativa è conservata: il primo introne interrompe la sequenza all’aminoacido 9, mentre il secondo introne interrompe la sequenza all’aminoacido 31 o 32, alla giunzione dei domini alpha e beta. CiMT-1 mostra la stessa struttura tripartita, con il primo introne nella posizione tipica dei vertebrati, mentre il secondo introne è posto nella regione 3‘UTR, come nelle MT degli echinodermi.
Anche la regione promotrice di CiMT-1mostra caratteristiche presenti sia negli echinodermi che nei vertebrati: possiede tre half-ARE (antioxidant responsive element) come negli echinodermi ed una sequenza ARE tipica dei vertebrati. Esso presenta inoltre quattro MRE (metal responsive elements) ben distanziati.
Il trattamento con Cd induce aumenti del trascritto di CiMT-1 con andamento irregolare: un primo aumento è presente a 6 h, seguito da una diminuzione fino a quasi i livelli di controllo a 24 h per poi ricrescere fino ad un picco massimo dopo 96 h. I trattamenti con Zn e Cu, invece, inducono valori di trascrizione quattro volte superiori rispetto ai livelli dei controlli solo a fine trattamento (120 h) con un andamento di crescita pressoché lineare nel tempo. Il trascritto per CiMT-1 è presente a livello dei soli granulociti. Tali cellule a 24 h di trattamento con Cd si accumulano nella tunica in numero circa doppio rispetto ai controlli. Pertanto la diminuzione di trascritto riscontrato a 24 h può essere conseguenza di questa migrazione cellulare, mentre il successivo aumento è presumibilmente da imputare a proliferazione cellulare specifica dei granulociti in circolo. Questo scenario è confermato dall’alto tasso di morte cellulare nella tunica, dove gli emociti si accumulano, ed inoltre dalla massiccia trascrizione di PCNA nei campioni trattati. Infatti sia CiMT-1 che PCNA presentano un picco di espressione a 96 h.. A confermare tale ipotesi sono i risultati relativi alla fitochelatino-sintetasi. In C. intestinalis il gene viene espresso dai granulociti ed i livelli di trascritto rimangono simili a quelli dicontrollo fino a 72 h, per poi crescere a 96 h. Da dati di letteratura è noto che questo gene non è inducibile, ma viene attivato in presenza di ioni metallici bivalenti. E’ molto probabile quindi, che questo picco non sia una conseguenza di un fenomeno di induzione, ma della forte proliferazione cellulare dei granulociti. E’ da notare che questo è il primo dato che dimostra la trascrizione di questo gene in organismi deuterostomi, la sua localizzazione esclusiva a livello dei granulociti e il suo coinvolgimento in processi di detossificazione da metalli.
Lo studio degli andamenti degli altri geni coinvolti nella detossificazione suggerisce che la componente enzimatica (SOD, GPX) possa avere un ruolo marginale, mentre la componente tiolica (PCS, GS, GCL) svolga un ruolo primario in presenza sia di Cd, che di Cu e Zn. Inoltre la localizzazione tramite ibridazione in situ di questi trascritti evidenzia che gli emociti circolanti sono le cellule maggiormente coinvolte nei processi di detossificazione
DIFFERENTIAL FORMS IN CARNOT GROUPS AFTER M. RUMIN: AN INTRODUCTION
No full textThese notes are taken from the Master Thesis of the second author
(written under the supervision of B. Franchi and P. Pansu) and are partially
based on a PhD course given by the first author at the University of Bologna
in 2012-2013. They are aimed to provide an elementary and comprehensive
introduction to the theory of differential forms in Carnot groups and to the
so-called Rumin’s complex
Preliminary characterization of complement in a colonial tunicate: C3, Bf and inhibition of C3 opsonic activity by compstatin
No full textThe complement system is a fundamental effector mechanism of the innate immunity in both vertebrates and invertebrates. The comprehension of its roots in the evolution is a useful step to understand how the main complement-related proteins had changed in order to adapt to new environmental conditions and life-cycles or, in the case of vertebrates, to interact with the adaptive immunity.
Data on organisms evolutionary close to vertebrates, such as tunicates, are of primary importance for a better understanding of the changes in immune responses associated with the invertebrate-vertebrate transition. Here we report on the characterization of C3 and Bf transcripts from the colonial ascidian Botryllus schlosseri (BsC3 and BsBf, respectively), a reliable model organism for immunobiological research, and present a comparative analysis of amino acid sequences of C3s and Bfs suggesting that, in deuterostomes, the structure of these proteins remained largely unchanged. We also present new data on the cells responsible of the expression of BsC3 and BsBf showing that cytotoxic immunocytes are the sole cells where the relative transcripts can be found. Finally, using the C3 specific inhibitor compstatin, we demonstrate the opsonic role of BsC3 in accordance with the idea that promotion of phagocytosis is one of the main function of C3 in metazoans
Morula cells as key hemocytes of the lectin pathway of complement activation in the colonial tunicate Botryllus schlosseri
No full textThe complement system is deeply rooted in the evolution of humoral mechanism of innate immunity. In
addition to the alternative pathway of complement activation, lectins and associated serine proteases
exert important roles in the recognition of non-self and activation of the effectors.
In the colonial tunicate Botryllus schlosseri, we identified, characterized and studied the expression of
three orthologues of genes involved in the lectin pathway of complement activation of vertebrates, i.e.,
genes for a mannose-binding lectin (MBL), a ficolin and a mannose-associated serine protease 1 (MASP1).
All the genes are transcribed by hemocytes, and specifically by morula cells, the same immunocytes
responsible for the transcription of C3 and Bf orthologues. The transcription levels of MASP1 and ficolin
orthologues are not affected by zymosan challenge, indicating a constitutive expression of complement
system associated serine proteases, whereas the MBL orthologue is up-regulated after 15 min of zymosan
exposure. Collectively, our data suggest the presence of a complete lectin activation pathway in Botryllus
Histamine stimulates ciliary beat frequency via the H2 receptor in the protochordate Botryllus schlosseri
No full textHistamine is a biogenic molecule that plays a role in many physiological pathways via binding to a specific receptor. Histaminergic receptors belong to the large family of seven-transmembrane α-helix domain receptors classified in mammals into four distinct classes: H1, H2, H3, and H4. Despite being widely studied in vertebrates, few data are available on the invertebrate receptors, with only predicted H1 and H2 sequences for nonchordate deuterostomes. Here, we report the first characterized transcript sequence for an H2 receptor from the colonial ascidian Botryllus schlosseri, describing the localization of both transcript and protein during blastogenic development through in situ hybridization and immunohistochemistry. Its phylogenetic relationships with deuterostome orthologous proteins are reported, its role in ciliary beat frequency (CBF) in cultured stigma cells of the branchial basket is outlined, and the effects of histamine and its receptor agonists and antagonists are analyzed. In the presence of increasing concentrations of histamine in the medium, CBF increases similarly to the selective H2 receptor agonist dimaprit. In contrast, ranitidine, which is an inhibitor of the H2 receptor, causes a significant inhibition of CBF, similar to that observed after preincubation with the specific anti-BsHRH2 or the anti-human HRH2 antibody. In cells bordering the branchial basket stigmata, both antibodies colocalize in the proximal region of the ciliary plasmalemma, and histamine is present inside vesicles of the apical region, thus supporting the hypothesis of a histamine-binding H2 receptor control of the pharyngeal mucociliary transport similar to that of the upper respiratory tract and middle ear in mammals
Immunity in Protochordates: The Tunicate Perspective
No full textTunicates are the closest relatives of vertebrates, and their peculiar phylogenetic position
explains the increasing interest toward tunicate immunobiology. They are filter-feeding
organisms, and this greatly influences their defense strategies. The majority of the studies
on tunicate immunity were carried out in ascidians. The tunic acts as a first barrier
against pathogens and parasites. In addition, the oral siphon and the pharynx represent
two major, highly vascularized, immune organs, where circulating hemocytes can sense
non-self material and trigger immune responses that, usually, lead to inflammation and
phagocytosis. Inflammation involves the recruitment of circulating cytotoxic, phenoloxidase
(PO)-containing cells in the infected area, where they degranulate as a consequence
of non-self recognition and release cytokines, complement factors, and the enzyme PO.
The latter, acting on polyphenol substrata, produces cytotoxic quinones, which polymerize
to melanin, and reactive oxygen species, which induce oxidative stress. Both the
alternative and the lectin pathways of complement activation converge to activate C3:
C3a and C3b are involved in the recruitment of hemocytes and in the opsonization of
foreign materials, respectively. The interaction of circulating professional phagocytes with
potentially pathogenic foreign material can be direct or mediated by opsonins, either
complement dependent or complement independent. Together with cytotoxic cells,
phagocytes are active in the encapsulation of large materials. Cells involved in immune
responses, collectively called immunocytes, represent a large fraction of hemocytes,
and the presence of a cross talk between cytotoxic cells and phagocytes, mediated by
secreted humoral factors, was reported. Lectins play a pivotal role as pattern-recognition
receptors and opsonizing agents. In addition, variable region-containing chitin-binding
proteins, identified in the solitary ascidian Ciona intestinalis, control the settlement and
colonization of bacteria in the gut
Influence of cadmium on the morphology and functionality of haemocytes in the compound ascidian Botryllus schlosseri
No full textIn order to get insights into the effects of cadmium (Cd) on cell morphology and functions, we exposed haemocytes of the colonial ascidian Botryllus schlosseri to sub-lethal concentrations of CdCl2. Results indicate that Cd hampers haemocyte spreading and phagocytosis in a dose-dependent way, through the alteration of the actin cytoskeleton. In addition, the metal decreases the stability of the internal membranes, as revealed by the Neutral Red assay. The fraction of cells showing positivity for the lysosomal enzyme acid phosphatase is also reduced in the presence of Cd, whereas the number of cells responsive to the Annexin-V assay and showing chromatin condensation increases, suggesting a metal-dependent induction of apoptosis in exposed cells. As Cd is a known cause of oxidative stress, the decrease in the percentage of cells positive to the assay
for superoxide anion, observed at low Cd concentrations, is indicative of the synthesis of metal-chelating molecules, such as metallothioneins, whereas, the increase at high Cd concentrations suggests a depletion of the cell reducing redox potential
Complement system receptors C3aR and CR1 in Tunicates: ancient roots of immunocyte dialogue.
No full textComplement system is one of the most important humoral defense mechanism of innate immunity. Its evolutionary history is rooted in cnidarians where C3 molecules have been described. C3 is considered, in all taxa, the main protein of the complement system and the peptides C3a and C3b, derived from its proteolysis, are the effectors of all the complement-related responses towards microorganisms. These peptides have been described in many taxa, but only in Vertebrates the complement system behaviour was deeply investigated, where more than 30 proteins were described. Conversely, in invertebrates, no more than 10 proteins in few organisms have been identified, all belonging to only two pathways of complement activation: the alternative and the lectin pathway.
Recently we focussed our attention on the complement system of the colonial ascidian Botryllus schlosseri. We identified C3 and the serine proteases associated with the alternative and lectin pathways. In the present work, we identified the main receptors of the activated C3: BsCR1 and BsC3aR. The former is the receptor of C3b; the latter of the anaphylotoxin C3a. The sequence and the structure of BsC3aR and BsCR1 are highly conserved. Both genes are constitutively transcribed; CR1 and C3aR are transcribed only in haemocytes, the former in circulating phagocytes and the latter in cytotoxic morula cells. .
The different expression sites of CR1 and C3aR indicates a similarity with what reported in vertebrates, where C3 can contribute to the activation of phagocytes, and what happen in ascidians, where C3 is produced by morula cells which, through the complement system, can orchestrate a complex dialogue with phagocytes during the immune response
New data on Botryllus schlosseri AMP: a transcript analysis
No full textThe innate immune system provides an immediate response against infections in animals and plants. Endogenous antimicrobial peptides (AMPs) are essential effector molecules of this first line of defence allowing the direct killing of invading micro-organisms. Moreover AMPs have attracted increasing interest because of their potential as new antibiotics.
To date various natural peptides from all kinds of organisms and synthetic derivatives have been characterized for their potential use as novel therapeutics. However, the number of candidate peptides undergoing preclinical or clinical evaluation is still very low. Marine invertebrates are source of several AMPs and some of these have been isolated and characterized from different urochordate such as Styela plicata (clavanins and styelins) and Ciona intestinalis (Ci-MAM).
In the colonial ascidian B. schlosseri, exploiting the transcriptome and the genome, we have been able to identify a styelin-like AMP. We also carried out some preliminary experiments to investigate the chemical properties and the expression pattern of such gene in the presence of different PAMPs
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