The search for the IFN-gamma receptor in fish: Functional and expression analysis of putative binding and signalling chains in rainbow trout Oncorhynchus mykiss

Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFN gamma R1 and IFN gamma R2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFN gamma Rs in fish. They are widely expressed in tissues, with IFN gamma R1 typically more highly expressed than IFN gamma R2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1 beta down regulating IFN gamma R1 expression but up regulating IFN gamma R2 expression. Overexpression of the two receptor chains in RTG-2 cells revealed that the level of IFN gamma R2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gamma IP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFN gamma R may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction. (c) 2009 Elsevier Ltd. All rights reserved.

Characterization of C-C chemokine receptor subfamily in teleost fish

Chemokines and their receptors play important roles in nervous and immune systems. Little information, however, exists concerning this gene family in teleost fish. In the present Study, 17 C-C chemokine receptors genes were identified from Danio rerio, 9 from Gasterosteus aculeatus, 10 from Oryzius latipes, 8 from Takifugu rubripes and 5 from Tetraodon nigroviridis. Phylogenetic analysis showed that the orthologs to mammalian CCR6, 7, 8, 9 and CCRL1 receptors were evident in zebrafish, but the clear orthologs to mammalian CCR1, 2, 3, 4, 5 and 10 were not found in zebrafish. The gene structure of zebrafish CCR (zfCCR) was further analyzed. The open reading frame of zfCCR3-1, zfCCR3-3, zfCCR6-1, zfCCR6-2, zfCCR8-2 contain one exon, and two exons were identified for zfCCR2-1, zfCCR2-2, zfCCR4 and zfCCRLI-1, three exons for zfCCR3-2, zfCCR5 and zfCCR7, four exons for zfCCR8-1 and zfCCR9-1. The expression analyses showed that in zebrafish, most C-C chemokine receptor genes Were expressed in fertilized eggs and oocytes, and all the receptor genes were expressed in larval stages. The zfCCR2-2, zfCCR3-1, zfCCR4 and zfCCR6-2 genes were expressed in all normal organs examined, whereas not for zfCCR2-1, zfCCR3-3, zfCCR6-1, zfCCR8-1, zfCCR9-2 and zfCCRL1-2. The expression of zfCCR3-2, zfCCR5, zfCCR7, zfCCR9-1 and zfCCRLI-1 were detected in the majority organs. and zfCCR8-2 and zfCCR8-3 detected only in brain. The differential expression pattern of different paralogues in organs may indicate their difference in function, which requires further investigation. (C) 2008 Elsevier Ltd. All rights reserved.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Characterization and expression analysis of TNFR-associated factor 1 (TRAF1) in grass carp Ctenopharyngodon idella

TNF receptor associated factor 1 (TRAF1) plays an important role in regulating the TNF signaling and protecting cells from apoptosis. In the present study, a TRAF1 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA is 2235 bp, including a 250 bp 5' UTR (untranslated region), a 1659 bp open reading frame, and a 326 bp 3'UTR. The polyadenylation signal (AATAAA, AATAA) and one mRNA instability motif (AUUUA) were found followed by a poly (A) tail in the 3'UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF1 (gcTRAF1). The putative amino acids of gcTRAF1 share 72% identity with the homologue in zebrafish. It is characterized by a zinc finger at the N-terminus and a TRAF domain (contains one TRAF-C and one TRAF-N) at the C-terminus. The identity of the TRAF domain among all the TRAF1 homologues in vertebrates varies from 52% to 58%, while the identities of TRAF-C were almost the same as 70%. The recombinant gcTRAF1 has been constructed successfully and expressed in Escherichia coli by using pET-32a expression vector. The polyclonal antibody for rabbit has been successfully obtained. The expression of gcTRAF1 in different organs was examined by real-time quantitative PCR and Western blotting, respectively. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of TRAF1 homologue molecule found in fish. (c) 2007 Elsevier B.V. All rights reserved.

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.

Subcellular localization and characterization of G protein-coupled receptor homolog from lymphocystis disease virus isolated in China

G protein-coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways, and play an important role in coordinating the activation and migration of leukocytes to sites of infection and inflammation. Viral GPCRs, on the other hand, can help the virus to escape from host immune surveillance and contribute to viral pathogenesis. Lymphocystis disease virus isolated in China (LCDV-C) contains a putative homolog of cellular GPCRs, LCDV-C GPCR. In this paper, LCDV-C GPCR was cloned, and the subcellular localization and characterization of GPCR protein were investigated in fish cells. LCDV-C GPCR encoded a 325-amino acid peptide, containing a typical seven-transmembrane domain characteristic of the chemokine receptors and a conserved DRY motif that is usually essential for receptor activation. Transient transfection of GPCR-EGFP in fathead minnow (FHM) cells and epithelioma papulosum cyprini (EPC) cells indicated that LCDV-C GPCR was expressed abundantly in both the cytoplasm and nucleoplasm. Transient overexpression of GPCR in these two cells cannot induce obvious apoptosis. FHM cells stably expressing GPCR showed enhanced cell proliferation and significant anchorage-independent growth. The effects of GPCR protein on external apoptotic stimuli were examined. Few apoptotic bodies were observed in cells expressing GPCR treated with actinomycin D (ActD). Quantitative analysis of apoptotic cells indicated that a considerable decrease in the apoptotic fraction of cells expressing GPCR, compared with. the control cells, was detected after exposure to ActD and cycloheximide. These data suggest that LCDV-C GPCR may inhibit apoptosis as part of its potential mechanism in mediating cellular transformation.

On the chemical synthesis and drug delivery response of folate receptor-activated, polyethylene glycol-functionalized magnetite nanoparticles

We describe here the chemical synthesis and in vitro drug delivery response of polyethylene glycol (PEG)-functionalized magnetite (Fe3O4) nanoparticles, which were activated with a stable ligand, folic acid, and conjugated with an anticancer drug, doxorubicin. The functionalization and conjugation steps in the chemical synthesis were confirmed using Fourier transform infrared spectroscopy. The drug-release behavior of PEG-functionalized and folic acid-doxorubicin-conjugated magnetic nanoparticles was characterized by two stages involving an initial rapid release, followed by a controlled release. (C) 2007 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.