124,204 research outputs found
Individuation of a new metallothionein from the urochordate Ciona intestinalis.
No full textMetallothioneins (MTs) are metal-chelating proteins occurring in animals, plants and prokaryotes, involved in detossification and immunity. In 2001, Canpolat and Lynes showed that exogenous MT can affect cell proliferation macrophage and cytotoxic T lymphocyte function, and humoral immunity to T-dependent antigens in mice. These evidences strengthen the hypothesis that MTs have an active role in extracellular environment as immunomodulatory proteins. Up to now, there are no descriptions of MTs in invertebrate Chordates althought it seems that the vertebrate structure is maintained also in other deuterostomes such as the echinoderms. As we are interested in studing the involvement and role of MTs ascidian immune responses, we undertook a preliminary investigation amied to identify these molecules in Ciona intestinalis, the genome of which has been fully sequenced but no MTs have been annotated. We have cloned the transcript and characterized the gene of a new MT, codifying for 39 amino acids, including 12 cys residues (30% of total amino acids, in accordance with other MTs). Moreover, the typical organization of cysteine residues in C-X-C motifs is conserved. The gene is composed of two introns (one inside the coding region and the other inside the 3’ UTR region) and three exons. The 5’ untrascribed region contains several cis elements similar to those found in vertebrate MT genes such as: metal responding elements (MRE) involved in constitutive and metal-related induction, antioxidant responding elements, important for ROS-dependent MT expression and STAT3, having a role in cytokine-related induction. The amino acid sequence of C. intestinalis MT shows only limited similarity with other known MTs: Mytilus edulis MT (28,8% identity), Strongylocentrotus purpuratus MT (23,4% identity) and Sparus aurata MT (36,7% identity)
The complement system of Botryllus schlosseri
No full textAmong the various effector mechanisms involved in immune responses, the complement system is one of the most ancient, deeply-rooted and important for its ability to orchestrate different cells and factors of both innate and adaptive immunity. The comprehension of its roots in the evolution is useful 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. In this context, data from 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 invertebratevertebrate transition. In our model tunicate Botryllus schlosseri we have described a lectin and alternative pathway of complement system activation very similar to those of Vertebrates. All the complement-related genes such as c3, bf, ficolin, mbl and masp are transcribed by morula cells, the immunocytes in immunomodulation and cytotoxic responses. Functional data suggest a complement-related cross-talk between morula cells and phagocytes immunocyte during the immune response. When B. schlosseri hemocytes are incubated with yeast (Saccharomyces cerevisiae) cells, there is an overexpression of C3 by morula cell that led to increase of phagocytosis that is prevented in the presence of the C3 inhibitor, compstatin. In the next future, we will focus our efforts on the regulation of complement system in tunicates to shed new light on the complement system function in a pre-adaptive immunity scenario
The C1q domain-containing protein from the ascidian Botryllus schlosseri manifests a cytokine-like behavior.
No full textGenes encoding complement component 3 (C3) have been extensively investigated in invertebrate genomes and traced back in evolutionary history to the early metazoan radiation. However, other components of the complement system, such as those related to the classical activation pathway, are still not much investigated. Currently, the genes encoding for proteins with a C1q domain, probably the main components of the classical pathway, have been only partially investigated from an evolutionary perspective. These genes exist in many of the sequenced genomes, from both vertebrates and invertebrates and functions have been described for some of the corresponding proteins. A C1q-like gene have been identified in the medicinal leech Hirudo medicinalis where a C1q-like peptide elicits a chemotactic behavior that could be blocked using a human antibody against the gC1q receptor. C1q-like genes have also been found in the urochordate Ciona intestinalis and the cephalocordate Branchiostoma floridae where it has been demonstrated that the globular domain is able to recognize and bind mammalian antibodies initiating the classical pathway of complement activation. The globular head C1q domain is a lectin domain present in transcriptomes of amphioxus, lamprey, and several teleost fishes. Few of these putative C1q-like proteins have been characterized; however, they can bind to a variety of carbohydrates. In Botryllus schlosseri we have found, in our EST collection, a single transcript with C1q characteristics (BsC1q-like). The deduced protein contains two globular head C1q domains, a feature unknown in invertebrates. As regard Vertebrates, we can find a similar architecture only in mammals, in the so called C1q/TNF-related Protein 4 (CTRP4). This protein is very poorly studied and seems to be expressed in the hypothalamus and contribute to the modulation of food intake and body weight. Our data, from the colonial ascidian, suggest a role for the BsC1q-like protein as mediator of the activation and degranulation of the cytotoxic hemocytes. Both ISH and ICC demonstrate that both cytotoxic morula cells and phagocytes express the BsC1q-like mRNA and protein; functional analyses demonstrate that the human antibody against globular head C1q is able to inhibit morula cell degranulation after bacterial challenge. It is not yet clear if it is possible to considered this molecule as member of the complement system in Botryllus but future analyses will be directed to the study of the functional relationships between BsC3 and BsC1q-like as well as of the binding capabilities of the latter
Evolution of the complement system: ancient molecules and new evidences from tunicates
No full textThe complement system is a key player in innate immunity but, recently, it is becoming even more evident that complement plays also important roles in adaptive immunity.
Components of the human complement system possess unique domain structures and are classified in protein families: C3, factor B (Bf), mannan-binding protein-associated serine protease (MASP), C6 and factor I (If) family.
These complement families probably derive from exon shuffling, which created the unique domain structures of each family, and gene duplication and subsequent functional divergence, which increased the number of members in each family. Accumulating information on the complement system of vertebrates indicates that these gene duplications, which played a pivotal role in establishing the classical and the lytic pathway, occurred in jawed vertebrates. In contrast, information on complement genes of invertebrate chordates is limited and, so far, only the ascidians, such as Ciona intestinalis and Halocinthia roretzi, and the cephalochordate Branchiostoma floridae have an almost complete set of the complement gene families: C3-, Bf-, MASP-, and C6-like genes.
In the present work we demonstrate the presence of C6-, C3-, MASP-, MBL- and Bf-like genes in the colonial ascidian Botryllus schlosseri and the enhanced transcription after zymosan infection, which indicates their involvement in the ascidian immunity. The observation of a transcribed C6 gene suggest that the presence of the lytic pathway predates the appearance of the vertebrates. We are now carrying out new investigation to demonstrate a complement-related lytic activity in B. schlosseri
Il volo da diporto o sportivo nel recente dPR 28 aprile 1993 n. 207
No full textL'articolo esamina le principali novità in materia di volo da diporto o sportivo introdotte dal dPR n. 207/1993
Preliminary studies of the complement system in Botryllus schlosseri.
No full textThe complement system represents an important humoral component of the mammalian immune system. Complement components can be subdivided in 5 gene families: C3/C4/C5, Bf/C2, MASP/C1r-s, C6/C7/C8A/C8B/C9 and Factor I.
Until 1884, it was generally believed that the complement system was an unique feature of vertebrates since all attempts to identify complement components in invertebrates failed. In recent years, the genomic approach revealed the presence of complement orthologue genes in invertebrate deuterostomes, mainly in sea urchins and tunicates (ascidians). Conversely, no complement genes were found in the genome of protostomes such as Drosophila melanogaster and Caenorhabditis elegans, suggesting that the complement system was established in the deuterostome lineage.
Genome analyses carried out in the solitary ascidian Ciona intestinalis revealed that most complement gene families are present in urochordates.
We recently carried out the assembling of EST collections from the colonial ascidian B. schlosseri, obtained in our and other laboratories: we found multiple transcripts showing high similarity with vertebrate complement components such as C3, MASP, MBL and C6. Preliminary in silico studies revealed close relationships between Botryllus C3, MASP and MBL and orthologues from other chordates. In particular, C6 seems related with the and Halocynthia roretzi and share with them the absence of the FIM domain which is responsible for the interaction with the other complement molecules in vertebrates.
Future studies will be devoted to the analysis of the expression of genes for complement components of B. schlosseri
New evidences of conserved pathways in complement system dynamics from the colonial ascidian Botryllus schlosseri.
No full textIn recent years, it has been widely demonstrated that complement, although often depicted as a ‘first line of defense’, is more than just a defender against microbial intruders and acts as a tightly integrated surveillance system. It is not only important against microorganisms, but also for the clearance of apoptotic cells and corpses. Botryllus schlosseri belongs, as vertebrates, to the phylum Chordata and, for this phylogenetic trait, it is unanimously considered a reliable model organism for the studies of the evolution of the immune system. Moreover it is also characterized by a peculiar life cycle with a cyclical, massive apoptosis. This two key features render B. schlosseri a good research tool for the study of the evolution of the complement system. Here we report the first results on the expression of BsC3 and BsFactorB, both components of the alternative pathway (AP) of complement activation, which form the AP C3 convertase.
Since studies on mammalian models have shown that 80 % of the observed complement response is derived from AP convertase-mediated C3 amplification, even if initially induced by the classical pathway (CP), studies of complement activation in an organism that lack the adaptive immunity, as B. schlosseri, could lead to a better comprehension of the AP cascade and the behavior of C3 convertase not only in invertebrates, but also in vertebrates, mammals included.
As in mammals, BsC3 is highly transcribed at basal level and over-expressed after incubation with non self (zymosan) while BsFactorB always shows limited expression. In the presence of compstatin, a 13-residue cyclic peptide able to inhibit the activation of C3 by C3 convertases, the percentage of phagocytosing hemocytes collapses. In the presence of both zymosan and compstatin, the transcription of BsC3 by hemocytes increases with respect to cells exposed only to zymosan: this suggests the presence of a conserved molecular machinery able to control and modulate B. schlosseri as well as the mammalian complement
Hemocyte proliferation influences metallothionein and phytochelatin levels in Ciona intestinalis.
No full textWe studied, through semiquantitative PCR and in-situ hybridization, the activities of the newly identified metallothionein and phytochelatin synthase genes from the solitary ascidian Ciona intestinalis (Ci-MT and Ci-PCS, respectively) in response to 10 μM CdCl2. Metallothioneins (MTs) and phytochelatins (PCs) are involved in detoxification systems of many organisms. An appreciable number of data are available for metazoan, especially for vertebrate, but very few data are available for urochordates. As the latter occupy the peculiar phylogenetic position of invertebrate chordates, the research on MTs and PCs in tunicates assumes a particular significance. Cd strongly induced Ci-MT, with a maximum at 4 days. Ci-PCS showed maximum expression at the same time. This result is probably related to a cell proliferation event, rather than an effective Ci-PCS gene activation. The hypothesis is supported by the strong induction of Proliferating Cell Nuclear Antigen (PCNA) transcript after 4 days of treatment and the colocalization in the hemocytes of the different riboprobes used. In addition, in literature is reported an expression profile similar to that of CiMT for C. intestinalis mannose binding lectins (CiMBL). Collectively, our data and data from the literature support the conclusion that hemocyte proliferation occurs in tunicate immune responses
Metallothioneins from urochordates: something new has happened.
No full textMetallothioneins (MTs) are able to bind essential and non-essential heavy metal ions, thus controlling cellular homeostasis and detossification. In addition, they act as scavengers for reactive oxygen species (ROS), thanks to their abundant thiols groups. They have a role also in the regulation of inflammatory responses through the modulation of immunomodulatory humoral components. Chordata represents the major phylum of Deuterostomes, including about 45,000 species distributed in three subphyla: Tunicata (Urochordata), Cephalochordata and Vertebrata. Invertebrate Chordata, about 3% of the total chordate species, are collectively named Protochordata. No MT genes have been annotated so far in Protochordata. In order to allow a comparison with the vertebrate MTs we undertook a search for MT genes in the genome of the solitary tunicate Ciona intestinalis. We were able to find a MT gene (CiMT1), which represents the first MT gene identified in Tunicates. Its expression is limited to haemocytes and modulated by Cd, Zn and Cu. The deduced protein is only 39 amino acids in length with no typical α and β domains. However the sequence shows that this protein shares the usual percentage ≥( 30) of Cys residues arranged in typical conserved motifs reported for vertebrates. We were able to identify this gene also in Molgula manhattensis and Ascidiella aspersa, the search in other tunicates gave no results. This data, with the evidence that in some urochordates is present the gene for a phytochelatin syntase, could suggest that other mechanisms of detossification are present in ascidians
Inchieste di sicurezza, indagini dell'Autorità giudiziaria: problematiche applicative del regolamento UE n. 996/2010
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