Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

The biological effects of rainbow trout (Oncorhynchus mykiss) recombinant interleukin-8

In this report, recombinant interieukin-8 (rIL-8) was produced and its activity tested for the first time in fish. The rainbow trout rIL-8 was produced in Escherichia coli and purified using a 6xHis tag at the N-terminus. The rIL-8 induced a dose-dependent migration of head kidney leukocytes at concentrations from 0.1 to 10 ng/ml, with a peak response at 1 ng/ml. Trout rIL-8 also had a significant effect on superoxide production by head kidney cells, with maximal, activity at 0.1 and 1 ng/ml. When injected intraperitoneally into trout, rIL-8 had a clear effect on total leukocyte number in the peritoneal cavity, with increasing doses (up to 5 mu g) eliciting more cells. Of three leukocyte types distinguished, neutrophils were the dominant cell type, especially at higher rIL-8 concentrations. In contrast, the proportion of macrophages and lymphocytes decreased with rIL-8 administration, suggesting that they were not attracted at the same rate as neutrophils. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of common carp (Cyprinus carpio L.) TCR gamma and CD3 gamma/delta chains

Partial cDNA sequences of TCR gamma and CD3 gamma/delta were isolated from the thymus of common carp (Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCR gamma and CD3 gamma/delta were obtained by means of 3' RACE and 5' RACE, respectively. The full length of carp TCR gamma chain is 1368 bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCR gamma contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCR gamma. The C region of carp TCR gamma contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR C gamma contains 37 amino acids. The full length of carp CD3 gamma/delta is 790 bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3 gamma/delta s, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3 gamma/delta. Differing from other known CD3 gamma/delta s, carp CD3 gamma/delta tacks the CXXCXE motif in the extracellular domain. RTPCR analysis demonstrated that the expression of TCR gamma gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCR gamma gene was detected in all the examined tissues. The expression of CD3 gamma/delta gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3 gamma/delta-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone. (c) 2007 Published by Etsevier Ltd.

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity. (C) 2007 Elsevier Inc. All rights reserved.

TRAIL in the mandarin fish Siniperca chuatsi: Gene and its apoptotic effect in HeLa cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the CYS231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPaLp, NF-kappa B and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis. (c) 2007 Elsevier Ltd. 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.

Molecular characterization and subcellular localization of Carassius auratus interferon regulatory factor-1

Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish but little is known about the relationship between its gene structure and nuclear 'ion of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. ouratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poty I:C, or CAB INF-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NILS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS Locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids (''(KDKSINK101)-K-95" and ''(75)KTWKANFR(82)"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K-75, K-78, R-82, K-95, and K-101) and one non-conserved basic amino acid (K-97) are present in this NLS from CaIRF-1. This observation suggests that K97 Of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic aminoacids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8 beta and CD4-like genes

Partial cDNA sequences of both CD8 beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8 and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8 is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8 beta s,carp CD8 beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8 beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8 beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp. (c) 2007 Published by Elsevier Ltd.

Induction and preliminary characterization of a novel halophage SNJ1 from lysogenic Natrinema sp F5

Halophage SNJ1 was induced with mitomycin C from Natrinema sp. strain F5. The phage produces plaques on Natrinema sp. strain J7 only. The phage has a head of about 67 nm in diameter and a tail of 570 nm in length and belongs morphologically to the family Siphoviridae. The phage is strongly salt dependent; NaCl concentration affects the integrity of SNJ1, phage adsorption, and plaque formation. The optimal NaCl concentration for phage adsorption and plaque formation is 30% and 25%, respectively.

Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection

Voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is acknowledged to play an important role in stress-induced mammalian apoptosis. In this study, Paralichthys olivaceus VDAC (PoVDAC) gene was identified as a virally induced gene from Scophthalmus Maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). The full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one alpha-helix, 13 transmembrane beta-strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells. This is the first report on the inductive expression of VDAC by viral infection, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI- 1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response. (c) 2007 Elsevier B.V. All rights reserved.

The neural signature of escalating frustration in humans.

Mammalian studies show that frustration is experienced when goal-directed activity is blocked. Despite frustration's strongly negative role in health, aggression and social relationships, the neural mechanisms are not well understood. To address this we developed a task in which participants were blocked from obtaining a reward, an established method of producing frustration. Levels of experienced frustration were parametrically varied by manipulating the participants' motivation to obtain the reward prior to blocking. This was achieved by varying the participants' proximity to a reward and the amount of effort expended in attempting to acquire it. In experiment 1, we confirmed that proximity and expended effort independently enhanced participants' self-reported desire to obtain the reward, and their self-reported frustration and response vigor (key-press force) following blocking. In experiment 2, we used functional magnetic resonance imaging (fMRI) to show that both proximity and expended effort modulated brain responses to blocked reward in regions implicated in animal models of reactive aggression, including the amygdala, midbrain periaqueductal grey (PAG), insula and prefrontal cortex. Our findings suggest that frustration may serve an energizing function, translating unfulfilled motivation into aggressive-like surges via a cortical, amygdala and PAG network.

Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

ISG15 is one of the most strongly induced genes upon viral infection, interferon (IFN) stimulation, and lipopolysaccharide, (LPS) stimulation, and only one copy has been found in mammals so far. Here two fish ISG15 genes, termed CaISG15-1 and CaISG15-2, have been cloned and sequenced from UV-inactivated GCHV (grass carp haemorrhagic virus)-infected and IFN-produced CAB cells (crucian carp Carassius auratus blastulae embryonic cells) by suppression subtractive hybridization. The full-length cDNA sequences of two crucian carp ISG15 encode a 155-amino-acid protein and a 161-amino-acid protein, both of which show 78.9% identity overall and possess the characteristic structures of mammalian ISG15 proteins including two tandem ubiquitin-like domains and the C-terminal canonical LRLRGG motif. In CAB cells treated with different stimuli including active virus, UV-inactivated GCHV and IFN containing supernatant (ICS), the expression of both CaISG15-1 and CaISG15-2 was up-regulated but displayed different kinetics. Poly I:C and LPS were also able to induce an increase in mRNA for both genes. In CAB cells responsive to active GCHV, UV-inactivated GCHV, CAB ICS, Poly 1:12 and LPS, CaISG15-1 was upregulated more significantly than CaISG15-2. These results suggest that there are two ISG15 homologues in crucian carp, both of which might play distinct roles in innate immunity against viral and bacterial infection. (c) 2006 Elsevier Ltd. All rights reserved.

Intelectin gene from the grass carp Ctenopharyngodon idella: cDNA cloning, tissue expression, and immunohistochemical localization

The cDNA encoding grass carp intelectin was isolated from a head kidney cDNA library, and termed gcIntL. The deduced amino acid sequence of gcIntL consists of 318 amino acids, and about 55% identical and 74% similar to human intelectin, which is a new type of lectin recognizing galactofuranose, and plays a role in the recognition of bacteria-specific components in animal hosts. The gcIntL gene consists of seven exons and six introns, spacing over approximately 3 kb of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcIntL formed a clade with Danio rerio intelectin and 35 kDa serum lectin. By real-time quantitative RT-PCR analysis, gcIntL transcripts were significantly induced in head kidney, trunk kidney, spleen, and intestine from LPS-stimulated fish. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcIntL gene have the same expression pattern, and both were detected in brain, gill, intestine, head kidney, trunk kidney, spleen, and heart. Furthermore, gcIntL protein could be detected in gill, intestine, trunk kidney, head kidney, spleen, heart, and brain including medulla oblongata and optic lobe, as determined by immunohistochemistry. This is the first report of intelectin expression pattern in fish, and of recombinant gcIntL and polyclonal antibody against gcIntL. (c) 2006 Elsevier Ltd. All rights reserved.

Short and long peptidoglycan recognition proteins (PGRPs) in zebrafish, with findings of multiple PGRP homologs in teleost fish

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (in: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response. (c) 2007 Elsevier Ltd. All rights reserved.