Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1)

Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system.     

Molecular characterization of nucleotide binding and oligomerization domain (NOD)-2, analysis of its inductive expression and down-stream signaling following ligands exposure and bacterial infection in rohu (Labeo rohita)

Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic pattern recognition receptor (PRR) and is a member of NOD like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products and is involved in innate immune responses. In this report, NOD-2 gene was cloned and characterized from rohu (Labeo rohita) which is highly commercially important fish species in the Indian subcontinent. The full length rohu NOD-2 (rNOD-2) cDNA comprised of 3176 bp with a single open reading frame (ORF) of 2949 bp encoding a polypeptide of 982 amino acids (aa) with an estimated molecular mass of 109.65 kDa. The rNOD-2 comprised two N-terminal CARD domains (at 4-91 aa and 111-200 aa), one NACHT domain (at 271-441 aa) and seven C-terminal leucine rich repeat (LRR) regions. Phylogenetically, rNOD-2 was closely related to grass carp NOD-2 (gcNOD2) and exhibited significant similarity (94.2%) and identity (88.6%) in their amino acids. Ontogeny analysis of rNOD-2 showed its constitutive expression across the developmental stages, and highlighted the embryonic innate defense system in fish. Tissue specific analysis of rNOD-2 by quantitative real-time PCR (qRT-PCR) revealed its wide distribution; highest expression was in liver followed by blood. In response to PGN and LTA stimulation, Aeromonas hydrophila and Edwardsiella tarda infection, and poly I:C treatment, expression of rNOD-2 and its associated downstream molecules RICK and IFN-γ were significantly enhanced in the treated fish compared to control. These findings suggested the key role of NOD-2 in augmenting innate immunity in fish in response to bacterial and viral infection. This study may be helpful for the development of preventive measures against infectious diseases in fish.     

Molecular cloning and functional characterization of porcine nucleotide-binding oligomerization domain-2 (NOD2)

The nucleotide-oligomerization domain (NOD) 2 is an important molecule involved in host defense. In this study, we report the cloning and characterization of porcine NOD2 (poNOD2) cDNA. The open reading frame of poNOD2 contains 3042 bp which encode 1013 amino acid residues. The putative poNOD2 protein shares higher level of homology with human counterpart (81.6% amino acid identity) than the mouse protein (76.6% amino acid identity). In order to determine the function of poNOD2, we established human embryonic kidney (HEK) 293 cells transfected to express poNOD2 cDNA. We found that poNOD2 was expressed not only in the cytoplasm but also in the inner side of the plasma membrane of HEK293 cells. HEK293 cells expressing poNOD2 responded to muramyl dipeptide (MDP) by activation of the nuclear factor kappa B (NF-kappaB). Quantitative real-time PCR revealed that poNOD2 mRNA was expressed by a number of tissues isolated from adult and newborn swine such as esophagus, duodenum, jejunum, ileum, ileal Peyer's patches (Pps), colon, spleen, and mesenteric lymph nodes (MLNs). In the newborn swine, the expression of poNOD2 mRNA was detected at higher levels in MLNs and spleen as compared to other tissues. In the adult swine, the highest expression was observed in ileal Pps. Furthermore, Toll-like receptor (TLR) and NOD2 ligands as well as immunobiotic lactic acid bacteria (LAB) enhanced the expression of NOD2 in gut-associated lymphoid tissues (GALT) in adult and newborn swine. Our results implicate NOD2 as an important immunoregulator in the swine intestinal immunity.     

Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand

We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016 bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-alpha and Lymphotoxin-alpha were 26.1%, 24.5% and 23.0%, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and introns is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system.     

Molecular cloning and functional characterization of porcine nucleotide-binding oligomerization domain-1 (NOD1) recognizing minimum agonists, meso-diaminopimelic acid and meso-lanthionine

In this study, we isolated a complementary DNA encoding nucleotide-binding oligomerization domain-1 (NOD1) from Peyer's patches (Pps) of swine gut-associated lymphoid tissues (GALT). The complete open reading frame of porcine NOD1 contains 2862 bp, encoding a 953-amino acid polypeptide. The porcine NOD1 amino acid sequence is more closely related to the human sequence (83.8% identity) than the mouse counterpart (79.2% identity). To examine the subcellular expression and function of porcine NOD1, we overexpressed it in human embryonic kidney 293 cells. Immunostaining with an anti-porcine NOD1 polyclonal antibody revealed that the protein was expressed in transfectants as an intracellular membrane-bound molecule. In the transfected cells, both gamma-d-glutamyl-meso-diaminopimelic acid, and meso-diaminopimelic acid and meso-lanthionine activated nuclear factor-kappa B. Quantitative real-time PCR detected NOD1 mRNA in multiple tissues isolated from adult and newborn swine, including the esophagus, duodenum, jejunum, ileum, ileal Pps, colon, spleen, and mesenteric lymph nodes. In the newborn and adults, NOD1 was highly expressed in the esophagus and GALT, such in the ileal Pps and mesenteric lymph nodes. Furthermore, Toll-like receptor and NOD1 ligands as well as immunobiotic lactic acid bacteria enhanced the expression of NOD1 in GALT of adult and newborn swine. Our results should help clarify how the intestinal immune system is modulated by low-molecular weight peptidoglycan fragments through NOD1.     

Orientia tsutsugamushi induced endothelial cell activation via the NOD1-IL-32 pathway

Orientia tsutsugamushi (OT), the causative agent of scrub typhus, is an obligate intracellular bacterium. In order to verify the inflammatory responses involved in the pathogenesis of scrub typhus, we assessed the cytokine profile of the human endothelial cell line, ECV304, after OT infection. We noted that CCL5, CCL17, IL-1α, IL-6, IL-8, IL-10, IL-15, TNF-α and TNF-β were strongly induced in response to OT. Additionally, IL-32, the candidate modulator for the induction of IL-6 and IL-8, was increased significantly with OT infection and these increases coincided with NOD1 pathway activation. Thus, we hypothesized that NOD1 pathway and IL-32 might act on cytokine release in endothelial cells as a modulator of the inflammation caused by OT infection. NOD1 siRNA resulted in a reduction in IL-32 levels, and also reduced IL-1β, IL-6, IL-8, and ICAM-1 expression in OT-infected ECV304 cells. These changes in IL-1β, IL-6, IL-8, and ICAM-1 induced by NOD1 knockdown were reversed as the result of IL-32 treatment. This indicated that OT infection activated the NOD1 pathway followed by IL-32 secretion, thus resulting in the production and expression of IL-1β, IL-6, IL-8, and ICAM-1. Therefore, IL-32 might perform a role upstream of the inflammatory reaction in endothelial cells of OT infection.     

Molecular cloning,expression analysis and functional characterization of interleukin-22 in So-iny mullet, Liza haematocheila

In the present study, interleukin-22 (IL-22) from So-iny mullet (Liza haematocheila) was identified, and its tissue expression in both healthy and Streptococcus dysgalactiae-infected fish was examined. The full length cDNA sequence of mullet IL-22 was 1070 bp, containing an open reading frame of 555 bp. The deduced amino acid sequence shared high similarity (45.1–67.9%) with IL-22 from other fish species. Mullet IL-22 also contained an IL-10 family signature and four cysteine residues that were well conserved in other vertebrate IL-22 molecules. Mullet IL-22 mRNA was highly expressed in kidney, moderately expressed in liver and gut, and relatively weakly expressed in spleen, and its expression was significantly up-regulated in all the examined tissues following S. dysgalactiae infection. Furthermore, recombinant mullet IL-22 protein was shown to promote the expression of β-defensin in the four tissues and to increase the survival rate of the fish infected with S. dysgalactiae. Our results suggest mullet IL-22 plays an important role in the immune defense against bacterial infection and has the potential to be used to treat bacterial diseases in fish.     

Seasonal variation and comparative analysis of non-specific humoral immune substances in the skin mucus of olive flounder (Paralichthys olivaceus)

The epidermal secretion of fish contains various non-specific immune substances that act as the first line of defense against invading pathogens. The present study investigated the level of mucosal antibodies, the activities of hemagglutinin and protease, and other enzymes in the skin mucus of farm reared olive flounder (Paralichthys olivaceus) for 1year, in order to gain an insight into the relationship between these mucosal immune substances and their seasonal variation. These levels varied significantly during different months of sample collection. The present study showed a positive correlation between water temperature and the level of mucosal antibodies, and an inverse relationship between the level of mucosal antibodies and the activity of mucosal hemagglutinin and protease, but no relationship between lysozyme activity and other innate immune substances. This relationship is thought to be a compensatory response in olive flounder to protect itself against pathogenic microorganisms which are inherently present in the aquatic environment.     

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.     

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 expression analysis of major histocompatibility complex class I,IIA and IIB genes of blunt snout bream (Megalobrama amblycephala)

Major histocompatibility complex (MHC) plays an important role in the immune response of vertebrates. In this study, we isolated MHC class IIA and IIB genes from blunt snout bream (Megalobrama amblycephala) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). In order to study the function of the MHC genes in M. amblycephala, tissue distribution and immune response of the MHC genes to bacterial challenge were analyzed. All the characteristic features of MHC class II chain structure could be identified in the deduced amino sequences of MHC IIA and IIB, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. The deduced amino acid sequence of the MHC IIA and IIB molecules shared from 48% to 88% and from 65% to 77% similarity with those of other teleosts, respectively. Quantitative real-time PCR (qRT-PCR) demonstrated that MHC I and II genes were ubiquitously expressed in ten tissues, with high level in immune related tissues, including kidney, intestine, gill and spleen. Challenge of M. amblycephala with the extracellular pathogen, Aeromonas hydrophila, resulted in a significant increase in the expression of MHC I, MHC IIA and IIB mRNA within 72 h after infection in gill, kidney, intestine and liver, followed by a recovery to normal level after 120 h. The changes of expression levels for MHC IIA and IIB in most tissues were significantly higher than that of MHC I in the corresponding tissues at most time points (P < 0.05). These results demonstrated the MHC genes played an important role in response to bacterial infection in M. amblycephala; however, MHC class I and II genes showed different functional activity, which need be further investigated in teleost.