Biomedical subjects

Tiehui Wang

Publications and source records attributed to Tiehui Wang.

8 recordsLinked to original sources

PubMed2005

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species.

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

Amino Acid Sequence↗

PubMed2005

Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss.

Interleukin (IL)-11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory effects. It was believed to be restricted to mammals, but here we report the first nonmammalian IL-11 gene, in rainbow trout (Oncorhynchus mykiss). A trout IL-11 cDNA clone was isolated that contains a 5'-untranslated region (UTR) of 400 bp, an open reading frame of 612 bp and a large 3'-UTR of 1924 bp. Analysis of a genomic DNA clone from a trout lambda library revealed that the trout IL-11 gene has the same five exon/four intron gene organization, as well as the same intron phase, as mammalian IL-11 genes. The 204 amino acid trout IL-11 translation has a predicted signal peptide of 26 amino acids and mature peptide of 178 amino acids, with a calculated molecular mass of 20.5 kDa and a theoretical pI of 9.83. The mature peptide contains a cysteine residue and a potential N-linked glycosylation site that are not present in mammals. Phylogenetic analysis clearly grouped trout IL-11 with IL-11 molecules from other species and separated from other members of the IL-6 family. The IL-11 gene is highly expressed in intestine and gills in healthy fish and its expression can also be detected in spleen, head kidney, brain, skin and muscle. Bacterial infection of rainbow trout markedly up-regulates IL-11 expression in liver, head kidney and spleen. IL-11 expression is also up-regulated in RTS-11 cells (a trout macrophage cell line), which constitutively expressed the lowest level of IL-11 of the four trout cell lines examined, after stimulation with bacteria, lipopolysaccharide, poly(I:C) and recombinant trout IL-1beta. Only a single transcript of 3.2 kb could be detected in lipopolysaccharide or recombinant IL-1beta-stimulated RNA samples by northern blotting. The expression results, showing that IL-11 is widely distributed and modulated by infection and other cytokines, suggest that fish IL-11 is an active player in the cytokine network and the host immune response to infection.

Amino Acid Sequence↗

PubMed2004

Sequencing and expression of the second allele of the interleukin-1beta1 gene in rainbow trout (Oncorhynchus mykiss): identification of a novel SINE in the third intron.

A lambda clone containing a rainbow trout IL-1beta1 gene was isolated by a PCR screening strategy from a genomic library cloned in lambda GEM-11, and an EcoRI fragment from this clone was fully sequenced, and contained 1680 bp 5'-flanking sequence, the whole IL-1beta1 gene open reading frame, and the 3'-flanking region with two potential poly A signals and poly A sites. This clone encoded a protein that shared 99.8% identity to the previously published trout IL-1beta1 cDNA sequence, with only three base substitutions. The main difference was that this clone had an additional complete HpaI SINE insertion in the 3rd intron making intron III 211 bp larger (834 bp via 623 bp). Thus this sequence was designated as allele B (Big intron III) of IL-1beta1 and the previously reported sequence as allele S (Short intron III). Three lines of evidence (allele specific PCR, cloning and sequencing, and direct sequencing of PCR products) revealed that allele B was constitutively expressed and could respond to stimulation with lipopolysaccharide or trout recombinant IL-1beta. Searching of the GenBank database with the HpaI SINE sequence resulted in three additional HpaI loci being identified in rainbow trout. Another SINE retroposition was also identified in the same intron of both alleles of IL-1beta1 by comparison with the trout IL-1beta2 gene. This novel SINE sequence, sharing high homology with the HpaI SINE at the 3'-end region, is present in EST databases of several species including human, mouse and fish. The consensus of this novel SINE shares 57 to 61% identities to tRNA-Leu from different species. Another older retroposition event in the same intron of IL-1beta1 has also been hypothesised, recognised as six adenines, that may function as a RNA polIII terminator. A model for the IL-1beta1 allele formation is proposed. Following the earliest retroposition into one of the two IL-1beta genes that resulted from a genome duplication in salmonids, the proper environment for successive PV SINE retroposition was created. A recent retroposition of the HpaI SINE in IL-1beta1 resulted in the formation of the two alleles of IL-1beta1. Examination of the SINEs insertion and their host gene microenvironments revealed that the SINE retroposition does not appear random, both in the site selection and the direction of insertion. The mechanism governing this outcome is discussed.

Alleles↗

PubMed2003

Complete sequencing and expression of three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway of the complement system in rainbow trout Oncorhynchus mykiss.

Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M(r) of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M(r) of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a alpha-chain of 773 aa, a beta-chain of 635 aa and a lambda-chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M(r) of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.

Amino Acid Sequence↗

PubMed2002

Cloning and functional characterisation of the interleukin-1 beta 1 promoter of rainbow trout (Oncorhynchus mykiss).

The upstream flanking region of the rainbow trout (Oncorhynchus mykiss) IL-1 beta 1 gene has been cloned and characterised functionally using luciferase-based reporter gene constructs, and the transcription start site (TSS) confirmed by RLM-RACE. A TATA box was present 27 bp upstream of the TSS, with an NF-kB site 19 bp upstream of the TATA box. Within 1217 bp of upstream sequence, 3 sites for NF-kB, 10 sites for NF-IL6, 15 sites for AP1, 6 sites for AP4, 2 sites for CHOP/CEBP alpha and 1 site for SP1 and PU.1 were identified. Seven potential sites for the transcription repressor Gfi-1 were also identified. Analysis of eight IL-1 beta 1s promoter luciferase constructs transfected into a trout fibroblast (RTG-2) cell line known to constitutively express IL-1 beta revealed that in the absence of intron 1, very low luciferase activity was detectable. All of the constructs containing intron 1 gave clear luciferase activity, with the highest luciferase activity detected with construct P2-4 containing 617 bp of upstream sequence. As little as 82 bp of upstream sequence gave relatively strong luciferase activity, a region containing both a PU.1 and NF-kB site. That NF-kB is a transcription factor required for expression of the trout IL-1 beta 1 gene was confirmed using inhibitor studies with lipopolysaccharide (LPS)-stimulated macrophages. Both trout recombinant IL-1 beta and LPS were able to increase luciferase activity in the reporter constructs, especially in those containing the most upstream sequence with the lowest constitutive expression. The possibility that an upstream repressor is functioning to inhibit constitutive expression of IL-1 beta in this species is discussed.

5' Flanking Region↗

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

Identification and analysis of an interleukin 8-like molecule in rainbow trout Oncorhynchus mykiss.

An interleukin 8 (IL-8) homologue has been identified in the rainbow trout Oncorhynchus mykiss. The transcript contains an open reading frame of 294 nucleotides that translates into a 97 amino acid putative peptide, with 5' and 3' untranslated regions (UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and flounder genes, the trout amino acid sequence lacks the typical ELR motif upstream of the first pair of cysteines, where DLR is present. The trout IL-8 gene contains four exons divided by three short introns of 341, 247 and 292bp, and occupies 1824bp of genomic DNA. RT-PCR reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear up-regulation of IL-8 expression, detectable by RT-PCR and Northern blot analysis.

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↗