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ABSTRACT: The SOCS proteins appear to define an important mechanism for the negative regulation of the cytokine-JAK-STAT pathway. In the present study, the mRNA expression profiles of a SOCS2 from Chinese mitten crab Eriocheir sinensis (EsSOCS2) after pentachlorophenol (PCP) treatment or RNA interference (RNAi) were analyzed to understand its possible regulatory roles in modulating the neurotransmitter release. The EsSOCS2 expression level in the PCP treated group was significantly higher than that of blank at 1.5, 3, 12 and 24 h after exposure, suggesting that EsSOCS2 might be involved in controlling and reducing neuronal cell damage resulted from PCP treatment. After the expression of EsSOCS2 gene was silenced by RNAi, the concentrations of catecholamines and nitric oxide (NO) were examined to evaluate the modulation of EsSOCS2 on the release of neurotransmitters. At 48 h after the treatment with sequence-specific dsRNA targeting EsSOCS2, the expression of EsSOCS2 was reduced to half compared to the original level, and the concentrations of norepinephrine and NO increased, while dopamine decreased significantly in haemolymph. The preliminary results indicated that EsSOCS2 regulated catecholaminergic neuroendocrine system to release catecholamines into haemolymph and might be an important feedback inhibitor of tyrosine kinase signaling pathways in crab, which subsequently regulated NO synthesis and prevented excessive NO release. This information is helpful to further understand the modulation of EsSOCS2 on neurotransmitter release in crab.
Fish & Shellfish Immunology 04/2013; · 3.32 Impact Factor
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ABSTRACT: Opioid growth factor receptor (OGFR) is a receptor for [Met5]-enkephalin and plays important roles in the regulation of cell growth and embryonic development. In the present study, a cDNA of 2,381 bp for the scallop Chlamys farreri OGFR (designated as CfOGFR) was identified by rapid amplification of cDNA ends (RACE) approach and expression sequence tag (EST) analysis. The complete cDNA sequence of CfOGFR contained an open reading frame (ORF) of 1,200 bp, which encoded a protein of 399 amino acids. The amino acid sequence of CfOGFR shared 33-64% similarity with other OGFRs. There was a low complexity domain and a conserved OGFR_N domain at the N-terminal of CfOGFR. The mRNA transcripts of CfOGFR were constitutively expressed in the tested tissues with the highest expression level in hepatopancreas. During the early embryonic development, the mRNA transcripts of CfOGFR could be detected in different development stages, where the expression level presented a downward trend as a whole. The stimulations of LPS, Glu and poly (I: C) significantly induced the expression of CfOGFR mRNA in haemocytes (P < 0.05), while PGN stimulation exerted no influence. Co-IP and western blot results revealed that the CfOGFR in haemocytes displayed high affinity and specificity to [Met5]-enkephalin. Exogenous [Met5]-enkephalin was observed to inhibit the proliferation of HEK293T cells transfected with pcDNA3.1(+)-CfOGFR in a time and dosage dependent manner. These results collectively indicated that CfOGFR, as a homologue of OGFRs in C. farreri, played an important role in cells proliferation, and might be involved in the immune response of scallops.
Fish & Shellfish Immunology 02/2013; · 3.32 Impact Factor
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ABSTRACT: The Rel/NF-κB transcription factors can function as key regulators to modulate the expression of immune-related genes in response to immune challenge or environmental stress. In the present study, a gene coding Rel/NF-κB homologue was identified from scallop Chlamys farreri (designated CfRel). Its deduced protein comprised 359 amino acids, and contained a conserved N-terminal Rel homology domain (RHD) and an IPT domain. There was an NF-κB/Rel/dorsal domain signature sequence in the RHD domain. The mRNA transcripts of CfRel could be detected in all the tested tissues including adductor muscle, mantle, gill, gonad, haemocytes, kidney and hepatopancreas, with the highest expression level in hepatopancreas. After LPS stimulation, there were two peaks of CfRel mRNA expression level in haemocytes at 6 h (25.25-fold, P < 0.05) and 24 h (59.66-fold, P < 0.05) respectively, while the mRNA expression of CfRel was only up-regulated at 3 h after PGN stimulation (2.35-fold, P < 0.05). By Western blotting technique, CfRel protein was observed in the cytoplasm and nucleus of scallop haemocytes, and its concentration in the haemocyte nucleus increased significantly at 3 h and 12 h after LPS stimulation. The noticeable NF-κB transcription activity of CfRel protein was determined by NF-κB luciferase reporter assays (122.43%, P < 0.05), and it decreased significantly (17.61%, P < 0.05) after the coexpression of scallop IκB protein. These results collectively suggested that CfRel mRNA transcripts and protein were induced by immune stimulation, and CfRel protein could extricate itself from IκB protein and transfer into the haemocyte nucleus to modulate the immune response in scallop.
Fish & Shellfish Immunology 02/2013; · 3.32 Impact Factor
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ABSTRACT: CpG oligodeoxynucleotides (CpG ODNs), also called bacterial DNA or synthetic oligodeoxynucleotides, can induce apparent immunity protection against various pathogens, and they are widely used as functional immunostimulant or vaccine adjuvant in mammals. In the present study, CpG-rich plasmid pUC57-CpG was constructed and employed to stimulate the shrimp Litopenaeus vannamei, and the total hemocyte count, percentage of apoptotic hemocytes, regeneration of circulating hemocytes, the ability of phagocytosis and generation of reactive oxygen species (ROS) were measured to reveal the possible protection mechanism of CpG ODNs. After the injection of pUC57-CpG, the total hemocyte count significantly decreased (p < 0.01) to 2.56 × 10(7) cell/mL at the first day post stimulation, while the apoptosis increased (p < 0.01), which was 1.72-fold of that in control group. At the same time, the regeneration of circulating hemocytes fluctuated in a similar trend, and a significant increase was observed at the first day post stimulation. The phagocytotic activity including the percentage of phagocytosis and phagocytotic index, experienced an upward tend during the whole experimental period and the ROS level increased by 22% (p < 0.05) compared to that in the control group at first day post stimulation. These results together suggested that pUC57-CpG could promote the apoptosis and regeneration of circulating hemocytes, and enhance the phagocytosis and ROS production, which might contribute to the boosted immunity against the infection of pathogens.
Fish & Shellfish Immunology 10/2012; · 3.32 Impact Factor
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ABSTRACT: Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3-6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg(-1) respectively, but it decreased to 10.82 U mg(-1) and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg(-1) in the present of trypsinase and Cu(2+). These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop.
Fish & Shellfish Immunology 05/2012; 33(2):375-81. · 3.32 Impact Factor
