Biomedical subjects

Chris J Secombes

Publications and source records attributed to Chris J Secombes.

10 recordsLinked to original sources

PubMed2006

Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3'-untranslated region of their mRNAs.

The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.

3' Untranslated Regions↗

PubMed2006

Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library.

The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.

Amino Acid Sequence↗

PubMed2005

The CD8alpha from sea bass (Dicentrarchus labrax L.): Cloning, expression and 3D modelling.

In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8alpha from sea bass (Dicentrarchus labrax L.). The sea bass CD8alpha cDNA is comprised of 1490 bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88 bp 5'-UTR and a 748 bp 3'-UTR. A multiple alignment of CD8alpha from sea bass with other known CD8alpha sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8alpha's. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8alpha grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8alpha sequences. The thymus was the tissue with the highest CD8alpha expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8alpha mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8alpha 11-120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten beta-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like beta-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S-S bond as seen in the template human and mouse structures.

Amino Acid Sequence↗

PubMed2004

Homologs of CD83 from elasmobranch and teleost fish.

Dendritic cells are one of the most important cell types connecting innate and adaptive immunity, but very little is known about their evolutionary origins. To begin to study dendritic cells from lower vertebrates, we isolated and characterized CD83 from the nurse shark (Ginglymostoma cirratum (Gici)) and rainbow trout (Oncorhynchus mykiss (Onmy)). The open reading frames for Gici-CD83 (194 aa) and Onmy-CD83 (218 aa) display approximately 28-32% identity to mammalian CD83 with the presence of two conserved N-linked glycosylation sites. Identical with mammalian CD83 genes, Gici-CD83 is composed of five exons including conservation of phase for the splice sites. Mammalian CD83 genes contain a split Ig superfamily V domain that represents a unique sequence feature for CD83 genes, a feature conserved in both Gici- and Onmy-CD83. Gici-CD83 and Onmy-CD83 are not linked to the MHC, an attribute shared with mouse but not human CD83. Gici-CD83 is expressed rather ubiquitously with highest levels in the epigonal tissue, a primary site for lymphopoiesis in the nurse shark, whereas Onmy-CD83 mRNA expression largely paralleled that of MHC class II but at lower levels. Finally, Onmy-CD83 gene expression is up-regulated in virus-infected trout, and the promoter is responsive to trout IFN regulatory factor-1. These results suggest that the role of CD83, an adhesion molecule for cell-mediated immunity, has been conserved over 450 million years of vertebrate evolution.

Amino Acid Sequence↗

PubMed2004

Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss.

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.

Animals↗

PubMed2004

Analysis of fish IL-1beta and derived peptide sequences indicates conserved structures with species-specific IL-1 receptor binding: implications for pharmacological design.

A large number of IL-1 protein sequences have become available recently from a range of vertebrate species and especially from bony fish. However, 3D structures are still only known for mammalian IL-1. In this review, we use a multiple sequence alignment of all published non-mammalian vertebrate IL-1beta proteins to locate the structurally important residues critical for maintaining the beta-trefoil fold and we investigate the degree to which functionally important residues involved in receptor binding are conserved across vertebrate species. We find that although there is a high level of variability of positions involved in receptor binding, the mode of binding and overall shape of the ligand-receptor complex is probably maintained. This implies that each species has evolved its own unique interleukin-1 signalling system through ligand-receptor co-evolution. Nonetheless, the IL-1beta processing mechanism in non-mammalian vertebrates remains unclear because, with the exception of three bony fish, all non-mammalian IL-1beta sequences discovered so far lack an ICE (Interleukin Converting Enzyme) cut site. The IL-1 system has become an important drug target because of its significance in inflammatory diseases. Research on peptides derived from IL-1beta has identified peptides that possess agonist activity in humans and in trout, and peptides with antagonist activity. The agonist peptides map to two distinct loop regions of IL-1beta that are known to interact with the flexible domain III of the corresponding receptor. Further analysis of the IL-1 system may prove useful in engineering IL-1 with improved features and in suggesting new avenues for therapeutic intervention.

Amino Acid Sequence↗

PubMed2003

Characterisation, expression and promoter analysis of an interleukin 10 homologue in the puffer fish, Fugu rubripes.

Using computer-based tools, an interleukin (IL) 10 homologue has been identified from the puffer fish ( Fugu rubripes) genome database. This is the first report on the existence of an IL-10 homologue in a non-mammalian vertebrate species. The Fugu IL-10 gene is located within a 2790-bp fragment including 549 bp of coding sequence which translates into an 183-amino-acid protein. It is predicted to contain five exons and four introns, sharing the same organization with the mammalian IL-10 genes. The size of the introns in the Fugu IL-10 gene is much smaller compared to mammalian IL-10 genes, whilst the size of the exons is similar. The deduced protein sequence shares 44-50% homology with the mammalian IL-10 sequences, 39-42% with the viral IL-10 sequences and 37-42% with other members of the IL-10 family, IL-20 and IL-22. Southern blot analysis indicates that a single copy of the IL-10 gene is present in the Fugu genome. A very low level of constitutive expression was detected in tissues of healthy fish including liver, kidney, gut and spinal cord, whilst no expression was detectable in spleen, gill, brain, gonad and eye. Analysis of the transcription regulation elements in the promoter region revealed that trans elements are located in the region between 1 bp and 721 bp, cis elements between 934 bp and 1114 bp and a tumour necrosis factor alpha responsive transcription element was located 92 bp upstream of the TATA box.

Amino Acid Sequence↗

PubMed2002

The first cytokine sequence within cartilaginous fish: IL-1 beta in the small spotted catshark (Scyliorhinus canicula).

Cartilaginous fish are considered the most primitive living jawed vertebrates with a complex immune system typical of all jawed vertebrates. Cytokine homologs are found within jawless and bony fish, although no cytokine or cytokine receptor genes have been sequenced in cartilaginous fish. In this study the complete coding sequence of the small spotted catshark (Scyliorhinus canicula) IL-1beta gene is presented that contains a short 5' untranslated region (54 bp), a 903-bp open reading frame, a 379-bp 3' untranslated region, a polyadenylation signal, and eight mRNA instability motifs. The predicted translation (301 amino acids) has highest identity to trout IL-1beta (31.7%), with greatest homology within the putative 12 beta-sheets. The IL-1 family signature is also present, but there is no apparent signal peptide. As with other nonmammalian IL-1beta sequences, the IL-1-converting enzyme cut site is absent. Expression of the IL-1beta transcript is detectable by RT-PCR in the spleen and testes, induced in vivo with LPS. Furthermore, a 7-fold increase of transcript levels in splenocytes incubated for 5 h with LPS was seen. The genomic organization comprises six exons and five introns with highest homology seen in exons encoding the largest amount of secondary structure per amino acid. Southern blot analysis suggests at least two copies of the IL-1beta gene or genes related to the 3' end of the IL-1beta sequence are present in the catshark. The cloning of IL-1beta in S. canicula, the first cytokine sequenced within cartilaginous fish, verifies previous bioactivity evidence for the presence of inflammatory cytokines.

Amino Acid Sequence↗

PubMed2002

Evolution of interleukin-1beta.

All jawed vertebrates possess a complex immune system, which is capable of anticipatory and innate immune responses. Jawless vertebrates possess an equally complex immune system but with no evidence of an anticipatory immune response. From these findings it has been speculated that the initiation and regulation of the immune system within vertebrates will be equally complex, although very little has been done to look at the evolution of cytokine genes, despite well-known biological activities within vertebrates. In recent years, cytokines, which have been well characterised within mammals, have begun to be cloned and sequenced within non-mammalian vertebrates, with the number of cytokine sequences available from primitive vertebrates growing rapidly. The identification of cytokines, which are mammalian homologues, will give a better insight into where immune system communicators arose and may also reveal molecules, which are unique to certain organisms. Work has focussed on interleukin-1 (IL-1), a major mediator of inflammation which initiates and/or increases a wide variety of non-structural, function associated genes that are characteristically expressed during inflammation. Other than mammalian IL-1beta sequences there are now full cDNA sequences and genomic organisations available from bird, amphibian, bony fish and cartilaginous fish, with many of these genes having been obtained using an homology cloning approach. This review considers how the IL-1beta gene has changed through vertebrate evolution and whether its role and regulation are conserved within selected non-mammalian vertebrates.

Amino Acid Sequence↗

PubMed2001

Cloning of iNOS in the small spotted catshark (Scyliorhinus canicula).

The first cartilaginous fish iNOS gene has been cloned in the small spotted catshark, Scyliorhinus canicula. The cDNA was 4568 bp long, with a 3375 bp open reading frame encoding a protein of 1125 amino acids and a predicted molecular mass of 127.8 kDa. The catshark translation had 77% amino acid similarity with chicken iNOS and 70-73% similarity with known teleost i NOS molecules. The various co-factor binding sites were well conserved, with the calmodulin site hydrophobicity profile noticeable more similar to tetrapod molecules than teleost molecules. The catshark iNOS transcript was not typically expressed constitutively, with the exception of the gills. Clear induction of the gene was seen in splenocytes after exposure to Vibrio anguillarum in vivo, and after stimulation with LPS in vitro. iNOS message was first seen 2 h after stimulation, and was still apparent 24 h post-stimulation, the last timing studies. Poly I:C was also able to induce iNOS transcript expression in splenocytes, albeit at a later timing (i.e.24 h). Such findings suggest a role for this molecule in the non-specific defences of cartilaginous fish as seen in other vertebrate groups.

Amino Acid Sequence↗