Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7 kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-κB and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-κB, as well as in regulating innate immune genes.     

Expression and functional characterization of PGRP6 splice variants in grass carp Ctenopharyngodon idella

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved pattern recognition receptors from insects to mammals, recognize bacterial PGN and function in antibacterial innate immunity. The existence of alternative splicing is a common feature for PGRP family. Here the splicing pattern from the splicing at the 5′ end of PGRP6 gene was identified in a teleost fish, the grass carp (Ctenopharyngodon idella). Four splice variants of grass carp PGRP6 were designated as gcPGRP6a, gcPGRP6b, gcPGRP6c and gcPGRP6d, respectively. Real-time PCR revealed the different expression of these variants in fish individuals and CIK cell line in response to stimulation with different microbial ligands. Immunofluorescence microscopy and Western blotting showed that the splice variants are intracellular protein. Cell lysates from Epithelioma papulosum cyprini (EPC) cells transfected with gcPGRP6 splice variants are able to bind microbial PAMPs including Lys-type PGN from Staphylococcus aureus, DAP-type PGN from Bacillus subtilis, glucan, mannan, and microorganisms including Streptococcus dysgalactiae, Flavobacterium columnare and Saccharomyces cerevisiae. Moreover, overexpression of gcPGRP6 variants inhibited earlier stage growth of intracellular bacteria. The data also identified a specific role for gcPGRP6c variant in the positive regulation of cytolytic molecule perforin, and for gcPGRP6a, gcPGRP6b and gcPGRP6c variants in positive regulation of antimicrobial peptides (AMPs). However, the gcPGRP6d variant, which encoded basically only the PGRP domain, failed to induce the expression of perforin and AMPs. It is suggested that fish PGRP6 splice variants have common and variant-specific function in innate immune response.     

Gig1 and Gig2 homologs (CiGig1 and CiGig2) from grass carp (Ctenopharyngodon idella) display good antiviral activities in an IFN-independent pathway

The virus-induced genes, Gig1 and Gig2, were identified first as IFN-stimulated genes (ISGs) from CAB cells. Previous studies suggested that Gig protein may have some potential antiviral functions. In this study, we cloned and identified the full-length cDNA sequences of Gig1 and Gig2 homologs (designated as CiGig1 and CiGig2, respectively) from grass carp (Ctenopharyngodon idella). The complete cDNA sequences of Gig1 and Gig2 contain 1231 bp and 690 bp, encoding for a 194 amino acid protein and a 158 amino acid protein, respectively. Their structure characteristics of CiGig1 and CiGig2 are highly similar to the corresponding homologues in crucian carp. The tissue-specific expressions of CiGig1 and CiGig2 in liver, spleen, kidney, intestine, gill and heart were significantly up-regulated following GCHV challenge. The results indicated that CiGig1 and CiGig2 may be involved in the antiviral immune responses in cells. To better understand the antiviral functions of CiGig1 and CiGig2 in vivo, CiGig1 or CiGig2 ORF cDNA were inserted into the plasmid pcDNA3.1, respectively. Subsequently, the recombinant plasmids were transfected into C. idellus kidney (CIK) cells. The over-expressions of CiGig1 and CiGig2 were observed in the CIK cells after treatment with GCHV. Cells with pcDNA3.1-CiGig1 or pcDNA-CiGig2 exhibited a relatively higher survival rate of (70.84% or 69.24%) than non-transfection (22.16%) and mock-vehicle controls (24.38%) following the virus infection. Our data showed that both CiGig1 and CiGig2 could exert antiviral effects effectively in vivo. Cycloheximide blocking protein synthesis demonstrated that both CiGig1 and CiGig2 mRNA expression could be induced by GCHV rather than by recombinant grass carp IFN (rCiIFN) directly, suggesting that CiGig1 and CiGig2 may not be IFN-stimulated genes since they display their antivirus activities in an IFN-independent pathway.     

Characterization of grass carp (Ctenopharyngodon idella) IL-17D: Molecular cloning,functional implication and signal transduction

Although the roles of IL-17 family members during inflammation have been extensively studied in mammals, their knowledge in lower vertebrates is limited. In particular, the biological activities of fish IL-17 and their functional roles are largely unknown. In this study, we cloned grass carp IL-17D (gcIL-17D) and found that its putative protein possessed the conserved features of IL-17 family members. Tissue distribution analysis showed that gcIL-17D was preferentially expressed in the mucosal tissues, including skin, gill and intestine. Subsequently, the involvement of gcIL-17D in inflammatory response was demonstrated by examining the expression profiles of gcIL-17D in head kidney and head kidney leukocytes following in vivo bacterial infection and in vitro LPS treatment, respectively. Furthermore, recombinant gcIL-17D (rgcIL-17D) was prepared in grass carp kidney cells and was able to promote the gene expression of some pro-inflammatory cytokines (IL-1β, TNF-α and CXCL-8) in grass carp primary head kidney cells, revealing gcIL-17D can function as a pro-inflammatory cytokine. Moreover, rgcIL-17D appeared to activate NF-κB signaling by modulating the phosphorylation of IκBα and up-regulated CXCL-8 mRNA expression possibly through NF-κB pathway. Our data shed new light on the functional role of teleost IL-17D in inflammatory response.     

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die-off of grass carp fingerlings and has therefore been called 'American grass carp reovirus' (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (>92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species 'Aquareovirus G' by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an 'evolutionary jump,' involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses.     

Gig1 and Gig2 homologs (CiGig1 and CiGig2) from grass carp (Ctenopharyngodon idella) display good antiviral activities in an IFN-independent pathway

The virus-induced genes, Gig1 and Gig2, were identified first as IFN-stimulated genes (ISGs) from CAB cells. Previous studies suggested that Gig protein may have some potential antiviral functions. In this study, we cloned and identified the full-length cDNA sequences of Gig1 and Gig2 homologs (designated as CiGig1 and CiGig2, respectively) from grass carp (Ctenopharyngodon idella). The complete cDNA sequences of Gig1 and Gig2 contain 1231 bp and 690 bp, encoding for a 194 amino acid protein and a 158 amino acid protein, respectively. Their structure characteristics of CiGig1 and CiGig2 are highly similar to the corresponding homologues in crucian carp. The tissue-specific expressions of CiGig1 and CiGig2 in liver, spleen, kidney, intestine, gill and heart were significantly up-regulated following GCHV challenge. The results indicated that CiGig1 and CiGig2 may be involved in the antiviral immune responses in cells. To better understand the antiviral functions of CiGig1 and CiGig2 in vivo, CiGig1 or CiGig2 ORF cDNA were inserted into the plasmid pcDNA3.1, respectively. Subsequently, the recombinant plasmids were transfected into C. idellus kidney (CIK) cells. The over-expressions of CiGig1 and CiGig2 were observed in the CIK cells after treatment with GCHV. Cells with pcDNA3.1-CiGig1 or pcDNA-CiGig2 exhibited a relatively higher survival rate of (70.84% or 69.24%) than non-transfection (22.16%) and mock-vehicle controls (24.38%) following the virus infection. Our data showed that both CiGig1 and CiGig2 could exert antiviral effects effectively in vivo. Cycloheximide blocking protein synthesis demonstrated that both CiGig1 and CiGig2 mRNA expression could be induced by GCHV rather than by recombinant grass carp IFN (rCiIFN) directly, suggesting that CiGig1 and CiGig2 may not be IFN-stimulated genes since they display their antivirus activities in an IFN-independent pathway.     

Dynamic localization and the associated translocation mechanism of HMGBs in response to GCRV challenge in CIK cells

High-mobility group box (HMGB) proteins, a family of chromatin-associated nuclear proteins, play amazingly multifaceted roles in the immune system of mammals. Thus far, little is known about the nucleocytoplasmic distribution of HMGBs in teleosts. The present study systematically investigated the dynamic localization of all six HMGB proteins in Ctenopharyngodon idella kidney (CIK) cells. Under basal conditions, all HMGBs exclusively localized to the nucleus. Grass carp reovirus (GCRV), polyinosinic–polycytidylic (poly(I∶C)) potassium salt and lipopolysaccharide (LPS) challenge evoked the nuclear export of HMGBs to various degrees: GCRV challenge induced the highest nuclear export of CiHMGB2b, and poly(I∶C) and LPS evoked the highest nucleocytoplasmic shuttling of CiHMGB1b. Overall, the nucleocytoplasmic shuttling of CiHMGB2a and CiHMGB3b was rarely induced by these challenges. Dynamic imaging uncovered that the nucleocytoplasmic GCRV-induced relocation of CiHMGB2b occurred in cells undergoing karyotheca rupture, apoptosis or proliferation. Western blot analyses were used to examine HMGB-EGFP fusion proteins in whole cell lysates, cytosol, nuclear fractions and culture medium. Further investigation demonstrated the nuclear retention of N-terminal HMG-boxes and the nucleocytoplasmic distribution of the C-terminal acidic tails. Comparative analyses of the dynamic relocation of full-length, truncated or chimeric HMGBs confirmed that the intramolecular interaction between HMG-boxes and C-tail domains mediated the nucleocytoplasmic translocation of HMGBs. These results not only provide an overall understanding of the subcellular localization of HMGBs, but also reveal the induction mechanism of the nucleocytoplasmic translocation of HMGBs by GCRV challenge, which lays a foundation for further studies on the interactions among pathogens, HMGBs and pattern recognition receptors in the innate immunity of teleosts.     

Association of the suppressor of cytokine signaling 1 (SOCS1) gene polymorphisms with acute coronary syndrome in Mexican patients

Recent studies provide evidence on the emerging role of the SOCS1 gene in the development and progression of atherosclerotic lesions. This gene encodes for the suppressor of the cytokine signaling-1 protein that interacts directly with the Janus kinases that are essential intracellular mediators of the immune cytokine action. The aim of this study was to test for associations between SOCS1 gene single nucleotide polymorphisms (SNPs) and the risk of developing acute coronary syndromes (ACS) in a group of Mexicans patients. Four SNPs [-3969 C > T (rs243327), -1656 G > A (rs243330), -820 G > T (rs33977706) and +1125 G > C (rs33932899)] of SOCS1 gene were determined for TaqMan genotyping assays in a group of 447 patients with ACS and 622 healthy controls. Under heterozygous model, the -3969 C > T (rs243327) SNP was associated with increased risk of ACS (OR = 1.45, P_Het = 0.021). On the other hand, under co-dominant and heterozygous models, the -1656 G/A (rs243330) SNP was associated with increased risk of ACS (OR = 1.47, P_Co-dom = 0.038 and OR = 1.50, P_Het = 0.013, respectively). Moreover, under co-dominant, dominant, and heterozygous models, the -820 T/G (rs33977706) SNP was associated with increased risk of ACS (OR = 1.59, P_Co-dom = 0.03, OR = 1.48, P_Dom = 0.028 and OR = 1.61, P_Het = 0.01). Finally, under co-dominant and heterozygous models, the +1125 G/C (rs33932899) SNP was associated with increased risk of ACS (OR = 1.54, P_Co-dom = 0.006, OR = 1.58, P_Het = 0.012, respectively). Models were adjusted for gender, age, body index mass, dyslipidemia, alcohol consumption, and smoking. In summary, our data suggests that the four studied polymorphisms of the SOCS1 gene play an important role as susceptibility markers for developing ACS.     

JK1 (FAM134B) represses cell migration in colon cancer: a functional study of a novel gene

Background

JK1 is a novel cancer-related gene with unknown functional role in carcinogenesis. The aim of this study is to investigate the role of JK1 gene in carcinogenesis in an in vitro cell proliferation and migration analysis model.

Methods

Small hairpin RNAs (shRNA) were designed to knock-down JK1 expression in colon cancer cell line (SW480) using transduction ready lentiviral particles. Cell proliferation and cell migration assays were performed on multiple extracellular matrices to investigate the cellular effects of JK1 in colon cancer cells. A non-cancer colonic epithelial cell line (FHC) was used to compare the expression of JK1 in cancer cell line.

Results

JK1 knock-down did not affect cellular proliferation or survival in colon cancer. However, the manipulation increased cancer cell migration rates on collagen and fibronectin substrates.

Conclusions

JK1 was shown for the first time to have a functional role in the pathogenesis of colon cancer. The results imply that JK1 represses the capacity of cancer cells to migrate within their tissue. They also concurred with the previous findings of JK1 activity correlations with clinical and pathological features in colon cancer. The capacity may have utility as a means to prevent cancer cells forming metastases.     

Molecular cloning and multifunctional characterization of GRIM-19 (gene associated with retinoid-interferon-induced mortality 19) homologue from turbot (Scophthalmus maximus)

GRIM-19 (gene associated with retinoid-interferon-induced mortality 19), a novel cell death regulatory gene, plays important roles in cell apoptosis, embryogenesis, mitochondrial respiratory chain and immune response. To date, little information is known about fish GRIM-19 characteristics except orange-spotted grouper (Epinephelus coioides). Here a new GRIM-19 gene is identified and characterized from turbot (Scophthalmus maximus), an economic marine fish in China and Europe. Briefly, turbot GRIM-19 is a 595-bp gene encoding a 144 amino acids protein, which shares the closest relationship with Atlantic halibut (Hippoglossus hippoglossus). The expression of turbot grim-19 in liver, spleen and kidney is up-regulated by the infection of Vibrio anguillarum and LCDV (lymphocystis disease virus). Subsequently, a recombinant protein of turbot GRIM-19 is acquired and the anti-bacterial function is proved by liquid culture inhibition experiment. The subcellular location indicates that turbot GRIM-19 is co-localized with STAT3 in the cytoplasm, which is mainly determined by GRIM-19 41–84 amino acids and STAT3 1–321 amino acids. Finally, the involvements of turbot GRIM-19 in cell apoptosis and NF-κB pathway are investigated. All these data help to understand GRIM-19 function in fish, as well as provide the application possibility of GRIM-19 in fish disease resistance breeding.     

Molecular cloning,expression and functional analysis of B-cell activating factor (BAFF) in yellow grouper, Epinephelus awoara

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation and immunoglobulin secretion. In this study, the yellow grouper (Epinephelus awoara) BAFF (designated EaBAFF) gene was cloned using RT-PCR and RACE (rapid amplification of cDNA ends) techniques. The full-length EaBAFF was 1442 bp and contained an open reading frame of 780 bp encoding a putative protein of 259 amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. The soluble BAFF (EasBAFF) had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-EasBAFF was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, the WST-8 assay indicated that EasBAFF was not only able to promote the survival/proliferation of yellow grouper splenic lymphocytes but also able to promote the survival/proliferation of mouse splenic B cells. Our findings may provide valuable information for research into the immune system of E. awoara and EasBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in fish.     

No association between high temperature requirement 1 (HTRA1) gene polymorphisms and Alzheimer's disease

High temperature requirement 1 (HTRA1) gene is a plausible risk factor in Alzheimer's disease (AD) as it encodes a protease known to degrade amyloid-β peptide. Here we have studied whether single nucleotide polymorphisms (SNPs) in the HTRA1 gene or its nearby regions associated with AD in a large clinic-based case-control cohort originating from Finland. We did not observe significant association of the HTRA1 SNPs with AD among the whole case-control cohort or age-at-onset risk effect among AD patients.     

Compartmentalized expression of light-induced clock genes in the suprachiasmatic nucleus of the diurnal grass rat (Arvicanthis niloticus)

Photic responses of the circadian system are mediated through light-induced clock gene expression in the suprachiasmatic nucleus (SCN). In nocturnal rodents, depending on the timing of light exposure, Per1 and Per2 gene expression shows distinct compartmentalized patterns that correspond to the behavioral responses. Whether the gene- and region-specific induction patterns are unique to nocturnal animals, or are also present in diurnal species is unknown. We explored this question by examining the light-induced Per1 and Per2 gene expression in functionally distinct SCN subregions, using diurnal grass rats Arvicanthis niloticus. Light exposure during nighttime induced Per1 and Per2 expression in the SCN, showing unique spatiotemporal profiles depending on the phase of the light exposure. After a phase delaying light pulse (LP) in the early night, strong Per1 induction was observed in the retinorecipient core region of the SCN, while strong Per2 induction was observed throughout the entire SCN. After a phase advancing LP in the late night, Per1 was first induced in the core and then extended into the whole SCN, accompanied by a weak Per2 induction. This compartmentalized expression pattern is very similar to that observed in nocturnal rodents, suggesting that the same molecular and intercellular pathways underlying acute photic responses are present in both diurnal and nocturnal species. However, after an LP in early subjective day, which induces phase advances in diurnal grass rats, but not in nocturnal rodents, we did not observe any Per1 or Per2 induction in the SCN. This result suggests that in spite of remarkable similarities in the SCN of diurnal and nocturnal rodents, unique mechanisms are involved in mediating the phase shifts of diurnal animals during the subjective day.