Biomedical subjects

Steve Bird

Publications and source records attributed to Steve Bird.

9 recordsLinked to original sources

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↗

PubMed2005

Identification and expression analysis of lymphotoxin-beta like homologues in rainbow trout Oncorhynchus mykiss.

A lymphotoxin-beta (LT-beta) gene has been cloned and sequenced in rainbow trout and provides the first conclusive evidence for the existence of LT-beta in teleost. Two isoforms of LT-beta were isolated. LT-beta1 cDNA was composed of 952 bp (with a 139 bp 5'-UTR and a 201 bp 3'-UTR) and LT-beta2 cDNA was 836 bp (with a 237 bp 5'-UTR and a 197 bp 3'-UTR) both of which translated into a protein of 203 amino acid residues. Both isoforms contained a predicted transmembrane domain of 21 amino acid residues (Leu11-Val31) and the TNF family signature (Val104-Phe120). Homology and phylogenetic analysis of trout LT-beta's with other known TNF family member showed good similarity to TNF-N (teleost) and other LT-beta (mammals and frog). LT-beta1 and TNF-alpha (1 and 2) genes were highly expressed in unstimulated trout head kidney, spleen, gill and intestine, whereas LT-beta2 was weakly expressed only in the gill. The expression of LT-beta1 and -beta2 genes was not found in macrophage (RTS-11) and fibroblast (RTG-2) like cell lines, although the TNF-alpha2 gene was expressed in both cell lines with the TNF-alpha1 gene only expressed in RTS-11 cells. In head kidney cells, expression of LT-beta1 and TNF-alpha (1, 2) genes was increased by stimulation with PHA or LPS. The discovery of trout LT-beta will allow a more complete analysis of fish inflammatory responses.

Adjuvants, Immunologic↗

PubMed2005

Characterisation and expression analysis of an interleukin 6 homologue in the Japanese pufferfish, Fugu rubripes.

The first IL-6 sequence in fish has been determined in Fugu rubripes by exploiting the synteny that is found between some regions of the human and Fugu genomes. The predicted 227 aa IL-6 homologue contains the IL-6/G-CSF/MGF motif, has a predicted secondary structure of four alpha-helixes but only contains two of the four cysteines important in disulphide bond formation. It shows low amino acid identities (20-29%) with known IL-6 sequences, although phylogenetic analysis groups the Fugu molecule with the other IL-6 molecules. The gene organisation of Fugu IL-6 and the level of synteny between the human and Fugu genomes has been well conserved during evolution with the order and orientation of the genes matching exactly to human chromosome 7. PHA stimulation of Fugu kidney cells resulted in a large increase in the Fugu IL-6 transcript, whereas LPS and Poly I:C resulted in a significant increase within spleen cells. The discovery of IL-6 in fish will now allow more detailed investigations of local inflammatory responses.

Amino Acid Sequence↗

PubMed2004

Characterisation and expression analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes, following their discovery by synteny.

This investigation provides the first conclusive evidence for the existence of the interleukin 2 (IL-2) and IL-21 genes in bony fish. The IL-2 and IL-21 sequences have been determined in Fugu rubripes by exploiting the conservation of synteny that is found between regions of the human and Fugu genomes. The predicted 149-amino acid IL-2 homologue contains the IL-2 family signature, has a predicted secondary structure of three alpha helixes and has the two cysteines important in disulphide-bond formation. It shows low amino acid identities (24-34%) with other known IL-2 sequences. The predicted 155-amino acid IL-21 homologue has a predicted secondary structure of four alpha helixes and has the four cysteines important in disulphide-bond formation. It shows low amino acid identities (29-31%) with other known IL-21 sequences. The gene organisation of Fugu IL-2 and IL-21 and the level of synteny between the human and Fugu genomes has been well conserved during evolution, with the order and orientation of the genes matching exactly to human Chromosome 4. Phytohaemagglutinin stimulation of Fugu kidney cells resulted in a large increase in the Fugu IL-2 and IL-21 transcripts. In vivo stimulation of Fugu with LPS and poly I:C showed IL-21 expression to be localised within mucosal tissues. The discovery of IL-2 and IL-21 in fish will now allow more detailed investigations into T-helper cell responses.

Amino Acid Sequence↗

PubMed2004

Mechanically braked Wingate powers: agreement between SRM, corrected and conventional methods of measurement.

In this study, we assessed the agreement between the powers recorded during a 30 s upper-body Wingate test using three different methods. Fifty-six men completed a single test on a Monark 814E mechanically braked ergometer fitted with a Schoberer Rad Messtechnik (SRM) powermeter. A commercial software package (Wingate test kit version 2.21, Cranlea, UK) was used to calculate conventional and corrected (with accelerative forces) values of power based on a resistive load (5% body mass) and flywheel velocity. The SRM calculated powers based on torque (measured at the crank arm) and crank rate. Values for peak 1 and 5 s power and mean 30 s power were measured. No significant differences (P >0.05) were found between the three methods for 30 s power values. However, the corrected values for peak 1 and 5 s power were 36 and 23% higher (P <0.05) respectively than those for the conventional method, and 27 and 16% higher (P <0.05) respectively than those for the SRM method. The conventional and SRM values for peak 1 and 5 s power were similar (P >0.05). Power values recorded using each method were influenced by sample time (P <0.05). Our results suggest that these three measures of power are similar when sampled over 30 s, but discrepancies occur when the sample time is reduced to either 1 or 5 s.

Anaerobic Threshold↗

PubMed2004

Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss).

A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41-45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1beta in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1beta in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1beta in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout.

Alternative Splicing↗

PubMed2003

Does lower-body BMD develop at the expense of upper-body BMD in female runners?

PURPOSE: Evidence suggests that exercise plays an important role in stimulating site-specific bone mineral density (BMD). However, what is less well understood is how these benefits dissipate throughout the body. Hence, the purpose of the present study was to compare the levels of, and the correlation between, BMD recorded at 10 sites in female endurance runners, and to investigate possible determinants responsible for any inter-site differences observed. METHODS: Repeated measures ANOVA was used to compare the BMD between sites and factor analysis was used to describe the pattern of intersite correlations. Allometric ANCOVA was used to identify the primary determinants of bone mass and how these varied between sites. RESULTS: The ANOVA and factor analysis identified systematic differences in BMD between sites, with the greatest BMD being observed in the lower-body sites, in particular the legs. An investigation into the possible mechanisms responsible for these differences revealed "distances run" (km.wk-1) as a positive, and "years of training" as a negative determinant of bone mass (P < 0.001). However, the effect of a number of determinants varied between sites (P < 0.05). Specifically, the ANCOVA identified that running further distances resulted in higher bone mass in the arms and legs. In contrast, training for additional years appeared to result in lower bone mass in the arms and lumbar spine. Calcium intake was also found to be positively associated with bone mass in the legs but negatively associated at all other sites. CONCLUSIONS: A combination of running exercise and calcium intake would appear to stimulate the bone mass of women endurance runners at lower-body sites but at the expense of bone mass at upper-body sites.

Adult↗

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↗