110 research outputs found
Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the lipopolysaccharide-challenged pharynx
The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ
hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of
proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were
shown to be upregulated 1 h post LPS challenge. The change in the expression of the
two Mif paralogs in the pharynx was assessed before and after stimulation, and qRTPCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters
of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis
Transforming growth factor β (CiTGF-β) gene expression is induced in the inflammatory reaction of Ciona intestinalis
Transforming growth factor (TGF-β) is a well-known component of a regulatory cytokines
superfamily that has pleiotropic functions in a broad range of cell types and is involved, in
vertebrates, in numerous physiological and pathological processes. In the current study, we report
on Ciona intestinalis molecular characterisation and expression of a transforming growth factor β
homologue (CiTGF-β). The gene organisation, phylogenetic tree and modelling supported the close
relationship with the mammalian TGF suggesting that the C. intestinalis TGF-β gene shares a
common ancestor in the chordate lineages. Functionally, real-time PCR analysis showed that
CiTGF-β was transcriptionally upregulated in the inflammatory process induced by LPS
inoculation, suggesting that is involved in the first phase and significant in the secondary phase of
the inflammatory response in which cell differentiation occurs. In situ hybridisation assays revealed
that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune
system, and expressed by cluster of hemocytes inside the pharynx vessels. These data supported the
view that CiTGF-β is a potential molecule in immune defence systems against bacterial infection
Evolution of Ciona intestinalis Tumor necrosis factor alpha (CiTNFα): Polymorphism, tissues expression, and 3D modeling
Although the Tumor necrosis factor gene superfamily seems to be very conserved in vertebrates, phylogeny, tissue expression, genomic and gene organization, protein domains and polymorphism analyses showed that a strong change has happened mostly in invertebrates in which protochordates were a constraint during the immune-molecules history and evolution. RT PCR was used to investigate differential gene expression in different tissues. The expression shown was greater in the pharynx. Single-nucleotide polymorphism has been investigated in Ciona intestinalis Tumor necrosis factor alpha (CiTNFα) mRNA isolated from the pharynx of 30 ascidians collected from Licata, Sicily (Italy), by denaturing gradient gel electrophoresis (DGGE). For this analysis, CiTNFα nucleotide sequence was separated into two fragments, TNF-1 and -2, respectively, of 630 and 540 bp. We defined 23 individual DGGE patterns (named 1 to 10 for TNF-1 and 1 to 13 for TNF-2). Five patterns for TNF-1 accounted for <10% of the individuals, whereas the pattern 13 of TNF-2 accounted for >20% of the individuals. All the patterns were verified by direct sequencing. Single base-pair mutations were observed mainly within COOH-terminus, leading to 30 nucleotide sequence variants and 30 different coding sequences segregating in two main different clusters. Although most of the base mutations were silent, four propeptide variants were detected and six amino acid replacements occurred within COOH-terminus. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure on COOH-terminus domain. Lastly we displayed the in silico 3D structure analysis including the CiTNFα variable region
Identification, cloning and environmental factors modulation of a ab defensin from the Lessepsian invasive mussel Brachidontes pharaonis (Bivalvia: Mytilidae).
Immunological effectors of invasive species playing a role in addressing new colonization are still poorly studied. In the present study the cDNA sequence of the defensin from a Lessepsian invasive species, the Red Sea mussel Brachidontes pharaonis, was cloned using RACE method. Defensins are a class of widely known antimicrobial peptides (AMPs), oligopeptides with a broad spectrum of targeted organisms ranging from viruses to parasites. Analysis of BpDef sequence (262 bp) revealed the presence of an ORF coding for 81 amino acids. The full-length amino acid sequence showed the highest similarity to antimicrobial peptides MGD1 and MGD2 sequence from Mytilus galloprovincialis. Phylogenetic analysis suggested that BpDef belongs to the αβ defensin AMPs with a typical domain structurally characterized by a α helix and two β sheets. BpDef mRNA is located in circulating hemocytes with small intra-cytoplasmic granules and with large granules. The transcription of defensin gene was modulated by the stress from temperatures and oxygenation condition. Temperatures of 20 °C did not stimulate a BpDef transcription over a short time. At 30 °C the kinetics of BpDef gene transcription showed up regulation after one day, while it was down regulated after six days, both under normoxia and hypoxia conditions
LPS Challenge Regulates Gene Expression and Tissue Localization of a Ciona intestinalis Gene through an Alternative Polyadenylation Mechanism
subtractive hybridization strategy for the identification of differentially expressed genes was performed between LPSchallenged
and naive Ciona intestinalis. This strategy allowed the characterization of two transcripts (Ci8short and Ci8long)
generated by the use of two Alternative Polyadenylation sites. The Ci8long transcript contains a protein domain with
relevant homology to several components of the Receptor Transporting Protein (RTP) family not present in the Ci8short
mRNA. By means of Real Time PCR and Northern Blot, the Ci8short and Ci8long transcripts showed a different pattern of
gene expression with the Ci8short mRNA being strongly activated after LPS injection in the pharynx. In situ hybridization
analysis demonstrated that the activation of the APA site also influenced the tissue localization of the Ci8short transcript.
This analysis showed that the Ci8long mRNA was expressed in hemocytes meanwhile the Ci8short mRNA was highly
transcribed also in vessel endothelial cells and in the epithelium of pharynx. These findings demonstrated that regulation of
gene expression based on different polyadenylation sites is an ancestral powerful strategy influencing both the level of
expression and tissue distribution of alternative transcripts
Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis
Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona
intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins.
In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried
out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory
responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis,
Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms
(Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina picta. The CiLgalsa
N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding,
whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable
as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on
CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD
were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling
process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgalsa
and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and
expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and
immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting
analysis indicated that CiLgals can form oligomer
Ciona intestinalis peroxinectin is a novel component of the peroxidase– cyclooxygenase gene superfamily upregulated by LPS
Peroxinectins function as hemoperoxidase and cell adhesion factor involved in invertebrate immune reac-
tion. In this study, the ascidian (Ciona intestinalis) peroxinectin gene (CiPxt) and its expression during the inflammatory response have been examined. CiPxt is a new member of the peroxidase–cyclooxygenase gene superfamily that contains both the peroxidase domain and the integrin KGD (Lys-Gly-Asp) binding motif. A phylogenetic tree showed that CiPxt is very close to the chordate group and appears to be the out-group ofmammalianMPO, EPO and TPO clades. The CiPxtmolecular structuremodel resulted superimposable to the human myeloperoxidase. The CiPxt mRNA expression is upregulated by LPS inoculation suggesting it is involved in C. intestinalis inflammatory response. The CiPxt was expressed in hemocytes (compartment/morula cells), vessel epithelium, and unilocular refractile granulocytes populating the inflamed tunic matrix and in the zones 7, 8 and 9 of the endostyle, a special pharynx organs homolog to the vertebrate thyroid glan
Phenoloxidases of different sizes are modulated by LPS inoculation into Ciona intestinalis tunic and pharynx
In the present study, to further characterize the pro-phenoloxidase (proPO) and active phenoloxidase (PO) involved in the Ciona intestinalis inflammatory response, tunic and pharynx homogenate supernatants were separated on high pressure liquid chromatography and fractions were assayed for the PO activity before and after LPS inoculation, as well as before and after trypsin treatment which activates proPO. The LPS inoculation per se did not significantly change the basal PO activity of the tunic homogenate supernatant (THS) and pharynx homogenate supernatant (PHS) restricted in two confluent peaks, whereas a significant enhancement was observable after the trypsin treatment. This trypsin effect suggests that proPO is the main component of the HPLC separated fractions, and indicates that LPS inoculation mainly challenges the pro-enzyme production by tunic cells and hemocytes, as well as the activation of the serine-protease pathway. The protein size analysis and DOPA-MBTH assay, disclose two active proteins of 90.0 and 170.0 kDa differently contained in the two main chromatographic peaks. Due to the SDS activating effect on the proenzyme analyzed by SDS-PAGE, the size of proPO could not be shown, whereas modulation of an oligomerization process of the 90 kDa component is suggested
Expression of a glucocorticoid receptor (D1GR1) in several tissues of the teleost fish Dicentrarchus labrax
Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization)
and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported.
Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity
leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of
DlGR1 localization are discussed
Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum
A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose-agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 degrees C, retained partial activity by treatment at 70 degrees C, and was fully inactivated at 90 degrees C. On western blot analysis, SauFBP showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, the similarity of the N-terminal sequence and a partial coding domain to teleost F-type lectins suggests that SauFBP32 is a member of this emerging family of lectins
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