查看更多>>摘要：Mammalian studies have shown that the nuclear transcription factor Y (NFYC) regulates the expression of major histocompatibility complex (MHC) by binding to CCAAT-box on promoters. However, few studies have focused on the regulatory mechanisms of NFYC in MHC pathway in fish. To explore the transcriptional regulatory mechanism of MHCIa in fish, we characterized NFYC and MHCIa of red-spotted grouper (Epinephelus akaara) (named EaNFYC and EaMHCIa, respectively). The EaNFYC genome sequence is 13,796 bp and contains 1,065 bp open reading frame. It is composed of ten exons and nine introns and encode a 354 amino acid sequence. The putative EaNFYC protein sequence shared 67.2-99.4% identity to vertebrate NFYC and possesses a typically conserved domain (histone- or haem-associated protein 5 domain (HAP5)) at the N-terminus. Transcripts of both EaNFYC and EaMHCIa were ubiquitously expressed in all detect tissues, and higher mRNA levels were detected in immune-relevant tissues (middle-kidney). EaNFYC expression increased after treatment with polyinosinic: polycytidylic acid, lipopolysaccharide, nervous necrosis virus, zymosan A, and Singapore grouper iridovirus. Analysis of subcellular localization indicated that EaNFYC was localized at the cell nucleus only. Furthermore, overexpression of EaNFYC significantly stimulated the expression of EaMHCIa, interferon signalling molecules and inflammatory cytokine. The region -878 bp to +82 bp of EaMHCIa promoter was identified to be the core promoter which EaNFYC take effect on. Additionally, point mutations and electrophoretic mobility shift assays verified that NFYC activate MHCIa expression by binding at the M1 and M2 binding sites that do not contain CCAAT-box. These results contribute to elucidating the function of fish NFYC on MHC transcriptional mechanisms, and provide the first evidence of positive regulation of MHCIa expression by NFYC in fish.
查看更多>>摘要：Clostridium perfringens (C. perfringens) type C (CPC) is one of the chief pathogens that causes diarrhea in piglets, and C. perfringens beta2 (CPB2) toxin is the main virulence factor of CPC. Our previous research demonstrated that ssc-microR-132 was differentially expressed in ileal tissues of CPC-mediated diarrheic piglets and healthy piglets, which implied a potential role of ssc-microR-132 in this process. Here, we found that ssc-microR-132 was notably down-regulated in CPB2-exposed intestinal porcine epithelial cells (IPEC-J2), which was consistent with the ileal tissue expression. Moreover, ssc-microR-132 upregulation alleviated CPB2-induced inflammatory damage and apoptosis in IPEC-J2, whereas ssc-microR-132 knockdown presented the opposite effects. Furthermore, the dual-luciferase reporter assay indicated that ssc-microR-132 directly targeted Dachshund homolog 1 (DACH1). Moreover, DACH1 overexpression intensified CPB2-induced inflammatory injury and apoptosis in IPEC-J2. Remarkably, the introduction of DACH1 weakened the anti-inflammatory and anti-apoptotic effects of ssc-microR-132 in CPB2-exposed IPEC-J2. Overall, the results reveal that ssc-microR-132 targeted DACH1 to alleviate CPB2-mediated inflammation and apoptosis in IPEC-J2.
查看更多>>摘要：Monitoring fish welfare has become a central issue for the fast-growing aquaculture industry, and finding proper biomarkers of stress, inflammation and infection is necessary for surveillance and documentation of fish health. In this study, a proteomic approach using mass spectrometry was applied to identify indicators of the acute response in Atlantic salmon blood plasma by comparing Aeromonas salmonicida subsp. salmonicida infected fish and non-infected controls. The antimicrobial proteins cathelicidin (CATH), L-plastin (Plastin-2, LCP1) and soluble toll-like receptor 5 (sTLR5) were uniquely or mainly identified in the plasma of infected fish. In addition, five immune-related proteins showed significantly increased expression in plasma of infected fish: haptoglobin, high affinity immunoglobulin Fc gamma receptor I (Fc gamma R1, CD64), leucine-rich alpha 2 glycoprotein (LRG1), complement C4 (C4) and phospholipase A2 inhibitor 31 kDa subunit-like protein. However, various fibrinogen components, CD209 and CD44 antigen-like molecules decreased in infected fish. Selected biomarkers were further verified by Western blot analysis of plasma and real time PCR of spleen and liver, including CATH1, CATH2 and L-plastin. A significant increase of L-plastin occurred as early as 24 h after infection, and a CATH2 increase was observed from 72 h in plasma of infected fish. Real time PCR of selected genes confirmed increased transcription of CATH1 and CATH2. In addition, serum amyloid A mRNA significantly increased in liver and spleen after bacterial infection. However, transcription of L-plastin was not consistently induced in liver and spleen. The results of the present study reveal novel and promising biomarkers of the acute phase response and inflammation in Atlantic salmon.
Omeka, W. K. M.Liyanage, D. S.Jeong, TaehyugLee, Sukkyoung...
9页
查看更多>>摘要：Superoxide dismutases (SODs) are metalloenzymes that convert superoxide radicals to H2O2 and O2. Although SODs have been extensively studied in mammals and other species, comparative studies in invertebrates, such as abalones, are lacking. Here, we aimed to characterize manganese superoxide dismutase in disk abalone (Haliotis discus discus) (AbMnSOD) by assessing its transcriptional levels at different embryonic developmental stages. Additionally, the temporal expression of AbMnSOD in different abalone tissues in response to bacterial, viral, and pathogen-associated molecular pattern (PAMP) stimuli was investigated. SOD activity was measured at various recombinant protein concentrations via the xanthine oxidase/WST-1 system. Cell viability upon exposure to H2O2, wound healing ability, and subcellular localization were determined in AbMnSOD-transfected cells. AbMnSOD was 681 bp long and contained the SOD-A domain. AbMnSOD expression was higher at the trochophore stage than at the other stages. When challenged with immune stimulants, AbMnSOD showed the highest expression at 6 h post-injection (p.i.) for all stimulants except lipopolysaccharides. In the gills, the highest AbMnSOD expression was observed at 6 h p.i., except for the Vibrio parahaemolyticus challenge. Recombinant AbMnSOD showed concentration-dependent xanthine oxidase activity. Furthermore, AbMnSOD-transfected cells survived H2O2-induced apoptosis and exhibited significant wound gap closure. As expected, AbMnSOD was localized in the mitochondria of the cells. Our findings suggest that AbMnSOD is an essential antioxidant enzyme that participates in regulating developmental processes and defense mechanisms against oxidative stress in hosts.
查看更多>>摘要：Sterile triploid fish (3n = 150), derived from the hybridization between red crucian carp (Carassius auratus red var., 9, 2n = 100) and allotetraploid (male, 4n = 200), exhibits the improved disease resistance compared with its parents, but the current knowledge of the immunity of triploid fish is limited. Here, we report the identification and characterization of melanoma differentiation-associated gene 5 (MDA5) homologues from red crucian carp, triploid fish and allotetraploid. In this study, one red crucian carp MDA5 transcript (2nMDA5), two triploid fish MDA5 transcripts (3nMDA5-a and 3nMDA5-b) and two allotetraploid fish MDA5 transcripts (4nMDA5-a and 4nMDA5-b) have been cloned and identified separately. Immunofluorescence staining assay displayed that these MDA5 proteins were cytoplasmic proteins. RT-qPCR assay showed that, in response to spring viremia of carp virus (SVCV) and poly (I:C) stimuli, the increase of 3nMDA5 mRNA level was obviously higher than those of 2nMDA5 and 4nMDA5. Interestingly, the reporter assay and plaque assay revealed collectively that 3nMDA5-b, a shorter splicing form of MDA5, exhibited the strongest IFN promoter-inducing ability and antiviral activity. Additionally, when co-expressed with 3nMAVS, 3nMDA5-b induced a considerably higher level of IFN promoter activation than 3nMDA5-a; and the interactions between 3nMAVS/3nMDA5-a and 3nMAVS/3nMDA5-b were verified by co-IP assay. Taken together, our findings support the conclusion that in triploid fish, 3nMDA5-b mediates a robust antiviral signaling in host innate immune response.
查看更多>>摘要：Toll-like receptors (TLRs) play a critical role in the innate immune response of fish. In this study, we isolated the cDNA sequence of Nile tilapia TLR1 (OnTLR1). The deduced OnTLR1 protein contains a signal peptide, 7 leucine-rich repeats (LRRs), a C-terminal LRR (LRR-CT), a transmembrane region and a highly conserved TIR domain. In healthy Nile tilapia, the OnTLR1 transcript was broadly expressed in all examined tissues, with the highest expression levels in the spleen. After infection with Streptococcus agalactiae, the OnTLR1 transcripts were upre-gulated in the gill and kidney. After stimulation with polyinosinic-polycytidylic acid (poly(I:C)), the expression levels of OnTLR1 were significantly downregulated in the intestine, whereas OnTLR1 transcripts were significantly upregulated in the kidney. After challenge with lipopolysaccharide (LPS), the expression levels of OnTLR1 were significantly upregulated in the spleen and kidney. The subcellular localization showed that OnTLR1 was expressed in the cytoplasm. TLR1 significantly increased MyD88-dependent NF-kappa B activity. However, the results of a pull-down assay showed that OnTLR1 did not interact with MyD88 or TIRAP. Binding assays revealed the specificity of OnTLR1 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C) and LPS. Taken together, these findings suggest that OnTLR1, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.
查看更多>>摘要：Stimulator of interferon gene (STING), an adaptor molecule in the immune system, is involved in mediating the response to viral and bacterial infections, anti-tumor immunity, autoimmune diseases, and lipid metabolism. There have been reports on the cloning and function of STING in humans, pigs, chickens, and cats; however, STING has not been characterized in non-human primates or monkeys to date. Therefore, in this study, the rhesus macaque (Macaca mulata) STING gene was cloned, and we performed preliminary functional tests to examine its role in the interferon (IFN) pathway. The M. mulatta STING complementary DNA was 1140 bp in length and encoded 380 amino acid residues. Phylogenetic analysis showed that Homo sapiens and M. mulatta STING are closely related and clustered on the same branch. M. mulatta STING was confirmed to increase the promoter activities of IFN-beta, nuclear factor-xB, and interferon-sensitive response element, and STING overexpression increased the mRNA levels of IFN-alpha, IFN-beta, and interferon regulatory factor 3. Infection of Marc-145 cells with porcine reproductive and respiratory syndrome virus activated STING, and its expression increased along with increases in viral multiplicity of infection titer and time. Moreover, STING expression was time-and dose-dependently up-regulated by poly (I:C) and poly (dA:dT) treatments in Marc-145 cells. In summary, these re-sults highlight STING as a vital immune system signal protein in the IFN pathway. This study provides a basis for understanding the immune characteristics of M. mulatta, and may have important implications for both monkey and human diseases.
查看更多>>摘要：Chicken has an impaired innate immune system compared with mammals. Some key innate immune genes, such as Retinoic acid-inducible gene I (RIG-I), Toll like receptor 8 (TLR8), Absent in melanoma 2 (AIM2) and IFN regulatory factor 3 (IRF3), are inactivated or missing due to DNA Insertion, gene partial deletion, or gene total deletion. A predicted N-terminal deleted chicken Cyclic GMP-AMP synthase (chcGAS) gene, which is proven as the most essential cytosolic DNA sensor in other species, be obtained from the GenBank database. The large fragment deletion makes the sequence accuracy and functional integrity of the predicted chcGAS open to dispute. Here, the exact chcGAS gene was first experimentally determined by 5 ' and 3 ' rapid amplification of cDNA ends (RACE) PCR, which specifically lacked 83 amino acids in the DNA binding domain. In addition, the conservation and feasibility of cGAS-STING signaling among different species were conducted by bioinformatics to explore the possibility of the existence of the conserved pathway in chickens. The basic characteristics of the chcGAS, such as macroscopic and microscopic distribution patterns of chcGAS have been studied. In order to better research the function of chGAS, a chcGAS knockout chicken cell line has been generated by CRISPR/CAS9. Together, chicken owns an N-terminal deleted cGAS gene, and more experimental evidences are urgently needed to verify the functional integrity of chcGAS.
查看更多>>摘要：Metallo-carboxypeptidases are exopeptidases with diverse expression and function, found in all kingdoms of life from bacteria to mammals. One of them, the carboxypeptidase A3 (CPA3), has become an important component of the mammalian immune system by its expression in mast cells. Mast cells (MCs) are highly specialized sentinel cells, which store large amounts of bioactive mediators, including CPA3, in very abundant cytoplasmic granules. Clinical studies have found an increased CPA3 expression in asthma but the physiological role as well as the evolutionary origin of CPA3 remains largely unexplored. CPA3 belongs to the M14A subfamily of metallocarboxypeptidases, which among others also includes the digestive enzymes CPA1, CPA2, CPB1 and CPO. To study the appearance of CPA3 during vertebrate evolution, we here performed bioinformatic analyses of homologous genes and gene loci from a broad panel of metazoan animals from invertebrates to mammals. The phylogenetic analysis indicated that CPA3 appeared at the base of tetrapod evolution in a branch closer to CPB1 than to other CPAs. Indeed, CPA3 and CPB1 are also located in the same locus, on chromosome 3 in humans. The presence of CPA3 only in tetrapods and not in fishes, suggested that CPA3 could have appeared by a gene duplication from CPB1 during early tetrapod evolution. However, the apparent loss of CPA3 in several tetrapod lineages, e.g. in birds and monotremes, indicates a complex evolution of the CPA3 gene. Interestingly, in the lack of CPA3 in fishes, zebrafish MCs express instead CPA5 for which the most closely related human carboxypeptidase is CPA1, which has a similar cleavage specificity as CPA3. Collectively, these findings clarify and add to our understanding of the evolution of hematopoietic proteases expressed by mast cells.
查看更多>>摘要：Interleukin-17 (IL-17) is a classic pro-inflammatory cytokine that plays an important role in the immune and inflammatory response. In the present study, the sequence feature of CgIL17-5 and its function as a pro inflammatory factor in inducing the mRNA expressions of downstream immune effectors were investigated in oyster Crassostrea gigas. There were two tightly folded alpha helixes and two pairs of antiparallel beta-pleated sheet in the amino acid sequence of CgIL17-5. The mRNA transcripts of CgIL17-5 were constitutively distributed in all the tested tissues, with the highest level in haemocytes. The mRNA expression level of CgIL17-5 in haemocytes increased significantly at 24 h after Vibrio splendidus stimulation. CgIL17-5 protein was mainly detected in granulocytes which were the main immunocompetent haemocytes in C. gigas. The phosphorylation of mitogen-activated protein kinases (CgJNK, CgERK and CgP38) and nuclear translocation of the transcription factors (CgRel and CgAP-1) in haemocytes were induced after the oysters received an injection of recombinant CgIL17-5 for 2 h. The mRNA expression levels of CgIL-17s, CgTNF-1, Cgdefh1 and Cgdefh2 increased significantly in haemocytes. At the same time, obvious branchial swelling and cilium shedding in gills were observed at 24 h after the oysters received an injection of rCgIL17-5. All the results collectively suggested that CgIL17-5 promoted the activation of CgMAPKs and the nuclear translocation of CgRel and CgAP-1 to promote the mRNA expressions of cytokines and antibacterial peptides.
NSTL
Elsevier