Francesco Buonocore

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Early treatment with Lactobacillus delbrueckii strain induces an increase in intestinal T-cells and granulocytes and modulates immune-related genes of larval Dicentrarchus labrax (L.).

Lactobacillus delbrueckii ssp. delbrueckii (AS13B), isolated from the gut of adult Dicentrarchus labrax, was administered live to developing sea bass using rotifers and Artemia as live carriers. Immune-related gene transcripts were quantified in post-larvae at day 70 post-hatch (ph) and histology, electron microscopy and immunocytochemistry of the intestinal tissue were performed at day 74 ph. Since the probiotic was orally administered the studies were focused on intestinal immunity. In treated fish gut integrity was unaffected, while the density of T-cells and acidophilic granulocytes in the intestinal mucosa was significantly higher than in controls. Probiotic-induced increases in intestinal T-cells and total body TcR-beta transcripts are first reported in fish. Significantly lower IL-1beta transcripts and a trend towards lower IL-10, Cox-2 and TGF-beta transcription were found in the treated group. Evidence is provided that early feeding with probiotic-supplemented diet stimulated the larval gut immune system and lowered transcription of key pro-inflammatory genes.

Purification and characterization of a veratryl alcohol oxidase enzyme from the lignin degrading basidiomycete Pleurotus ostreatus

A veratryl alcohol oxidase (VAO) enzyme was discovered in cultures of Pleurotus ostreatus. The enzyme, which oxidizes veratryl alcohol to veratraldehyde reducing O2 to H2O2, was purified to homogeneity and its main structural and catalytic properties have been determined. The enzyme is a glycoprotein and contains FAD as a prosthetic group. The amino acid composition and carboxy- and amino-terminal sequences were determined. Primary aromatic alcohols with methoxy substituents in position four are good substrates for VAO; cinnamyl alcohol is the substrate which is oxidized faster whereas coniferyl alcohol is oxidized at a slower rate. The enzyme is moderately thermostable (t1/2(55 degrees C) about 1.5 h, apparent melting temperature about 60 degrees C). The enzyme stability in 50% water/organic solvents mixtures has also been studied.

Biological Activity of Sea Bass (Dicentrarchus labrax L.) Recombinant Interleukin-1β

Biological activities of a putative mature sea bass interleukin-1β peptide, produced as a recombinant protein (rIL-1β) in Escherichiacoli, have been investigated. The rIL-1β contains a 6-histidine tag at the N-terminus, and protein purification has been achieved through this tag by affinity chromatography. Biological activities have been investigated both at the cellular and gene expression levels. In in vitro assays sea bass rIL-1β induced the proliferation of murine D10.G4.1 cells and increased yeast phagocytosis by sea bass head kidney leukocytes. The purified cytokine was also tested in a lymphocyte-activation factor assay, where it induced the proliferation of sea bass thymocytes. Finally, in an in vivo assay, rIL-1β administered intraperitoneally increased expression levels of the IL-1β gene and activated macrophages to produce a cyclooxygenase 2 homologue (COX-2) gene in the head kidney.

Immunoglobulin protein and gene transcripts in ovarian follicles throughout oogenesis in the teleost Dicentrarchus labrax

Transfer of immunoglobulins (IgM-like) from the female to the teleost embryo has been demonstrated but mechanisms of uptake into and storage within the eggs remain to be clarified. The monoclonal antibody DLIg3 against Dicentrarchus labrax Ig light chain revealed an active role of both follicle cells and oocytes in the Ig uptake. The primordial follicular cells showed DLIg3 immunoreactivity even at a pre-vitellogenetic stage. Early vitellogenetic oocytes (lipid vesicle stages) had DLIg3 staining of pore canals, plasmalemma and outer cortex and of their follicular cells. In protein yolk granule oocytes, DLIg3 staining was also detected within vesicles of the outer-mid cortex and juxtanuclear yolk granules; therefore, a centripetal transport of Ig throughout oocyte development is apparently carried out. Immunoelectron microscopy confirmed the presence of Ig within thecal and granulosa cells (and in the interposed basement membrane) of pre-vitellogenic and vitellogenic follicles. Thus, the transport of Ig to the egg apparently occurs also by transcytosis across the follicle cells. Igs were localised in the pore canals surronding the microvilli and in vesicles of outer-mid cortex of vitellogenic oocytes. Reverse transcription/polymerase chain reaction with primers designed for the constant region of sea bass Ig light chain detected Ig mRNA in hydrated oocytes, a smaller content in released eggs and no signal in larvae at day two post-hatching. These findings show that a significant level of Ig gene transcription in the oocyte and/or a transfer of transcripts may also occur.

The immune system of sea bass, Dicentrarchus labrax, reared in aquaculture.

The sea bass Dicentrarchus labrax is one the most important seawater fish species of south Europe and Mediterranean aquaculture, and studies on its immune system are important for both scientific and applied purposes. In this paper, we summarise the results obtained in studies of the immune system in this species, and present original data on cell-mediated acquired immune response.

A CD4 homologue in sea bass (Dicentrarchus labrax) : Molecular characterization and structural analysis

CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell co-receptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4h and a decrease after 24h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals.

Expression in Escherchia coli and Purification of Sea Bass (Dicentrarchus labrax) Interleukin 1β, a Possible Immunoadjuvant in Aquaculture

AbstractInterleukin 1β (IL-1β) is a pleiotropic cytokine that plays a pivotal role in regulating immune responses. Our group has recently cloned IL-1β from sea bass (Dicentrarchus labrax), one of the main Mediterranean aquacultured fish species. The cDNA is 1292 bp and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. As for trout and carp IL-1β precursor sequence, no candidate cut site for ICE (IL-1β converting enzyme) enzyme was apparent in the alignments of sea bass IL-1β with other mammalian IL-1βs. Nevertheless, a possible mature peptide could start at Ala86, giving a protein of 176 amino acids. The nucleotide sequence coding for this polypeptide was cloned into a pQE-30 expression vector. The plasmid was then transformed in Escherichia coli, and the recombinant protein was purified. Finally, we demonstrated that this purified recombinant IL-1β was able to induce IL-1β gene expression in a dose-dependent manner on cells purified from sea bass head kidney and could have immunoadjuvant effects in sea bass vaccination experiments.

Transcription of T cell-related genes in teleost fish, and the European sea bass (Dicentrarchus labrax) as a model

In recent years the cloning of genes coding for immuno-regulatory peptides, as well as the sequencing of genomes, provided fish immunologists with a growing amount of information on nucleotide sequences. Research is now also addressed in investigating the functional immunology counterpart of nucleotide sequence transcripts in various fish species. In this respect, studies on functional immunology of T cell activities are still at their beginning, and much work is needed to investigate T cell responses in teleost fish species. In this review we summarise the current knowledge on the group of genes coding for main T cell-related peptides in fish, and the expression levels of these genes in organs and tissues. Particular attention is paid to European sea bass (Dicentrarchus labrax), a marine species in which some information on functional immunology has been obtained, and we reassume here the expression of some T cell-related genes in basal conditions. In addition, we provide original data showing that T cells purified from the intestinal mucosa of sea bass with a specific mAb, express transcripts for TRβ, TRγ, CD8α, and RAG-1, thus showing similarities with intra-epithelial leucocytes of mammals.

A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths

The recent availability of both robust transcriptome and genome resources for coelacanth (Latimeria chalumnae) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods.