Molecular cloning, characterization and expression analysis of the Chinese soft-shelled turtle (Pelodiscus sinensis) chemokine CXCL11.

Chemokines are small, secreted proteins with chemoattractive properties, which play an important role in the recruitment and activation of immune cells. CXCL11 is a CXC chemokine specific for the CXCR3 receptors, which has been shown to mediate the generation of Th1-type immune responses and have bactericidal effects similar to defensins. Herein, we cloned the full-length cDNA of Chinese soft-shelled turtle (Pelodiscus sinensis) CXCL11, designated as PsCXCL11, which consist of an open reading frame (ORF) of 282 bp encoding 93 amino acids, with estimated molecular weight of 10.055 kDa and isoelectric point of 10.37. The deduced PsCXCL11 sequence had a signal peptide, a highly conserved family-specific small cytokine (SCY) domain, one putative N-glycosylation site and ten potential phosphorylation sites. Phylogenetic analysis showed a close relationship between P. sinensis and Chelydra Serpentina CXCL11. P. sinensis CXCL11 basal expression levels were higher in heart, kidney and spleen than in other organs of health turtles. Infections of Aeromonas hydrophila and Staphylococcus aureus led to significant upregulation of P. sinensis CXCL11 in the blood, while significant upregulation of PsCXCL11 were observed in liver and spleen after infection of A. hydrophila, but not S. aureus. PsCXCL11 recombinant protein with His-tag was successfully expressed by an auto-inducible expression system, and purified by Ni-NTA affinity chromatography. These findings laid a solid foundation for further research towards development of the Chinese soft-shelled turtle as a model for the role of CXCL11 in regulating inflammatory responses to stimulation by invading pathogens.

Identification, characterization and expression analysis of ERK2 in Chinese mitten crab Eriocheir sinensis after challenge with LPS and Aeromonas hydrophila.

Farming of Eriocheir sinensis was seriously threaten by the infection of opportunistic pathogens, especially the gram-negative bacterium. In this paper, we analyzed the sequence of extracellular signal-regulated kinases 2 (ERK2) of E. sinensis (EsERK2) and its expression levels after challenge with LPS and Aeromonas hydrophila in both in vivo and in vitro examination. The full-length cDNA sequence of EsERK2 was 2455 bp in size with an open reading frame (ORF) of 1095 bp, encoding the protein of 365 amino acids. It owned a predicted molecular mass of 42.4 kDa and a theoretical isoelectric point (pI) of 5.93. EsERK2 was distributed in all examined tissues including haemocyte, gonad, hepatopancreas, gill, muscle heart, stomach and intestine, but its expression level was significantly higher in hepatopancreas than it in other examined tissues. The expression level of EsERK2 increased significantly after LPS challenge at 2 h (P < 0.05), and then gradually increased and reached highest at 16 h. However, its expression level decreased significantly after A. hydrophila challenge at 4 h, and then gradually decreased till 24 h (P < 0.05), and returned to its initial value at 36 h. According to the immunofluorescence assay and western blotting assay, EsERK2 was found to be distributed mainly in cytoplasm of haemocyte, and its expression level showed a prominent boost in primary cultured haemocytes after challenge with LPS and A. hydrophila in vitro. These results indicated that the expression of EsERK2 was sensitive to the exterior stimulants and its encoding protein might be associated with the signaling transduction in response to exterior pathogens in E. sinensis.

Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis.

Pelodiscus sinensis is the most common turtle species that has been raised in East and Southeast Asia. However, there are still limited studies about the immune defense mechanisms in its small intestine until now. In the present research, histological analysis and transcriptome analysis was performed on the small intestine of P. sinensis after intragastric challenge with LPS to explore its mechanisms of immune responses to pathogens. The result showed the number of intraepithelial lymphocytes (IELs) and goblet cells (GCs) in its intestine increased significantly at 48 h post-challenge with LPS by intragastrical route, indicating clearly the intestinal immune response was induced. Compared with the control, a total of 748 differentially expressed genes (DEGs) were identified, including 361 up-regulated genes and 387 down-regulated genes. Based on the Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG), 48 immune-related DEGs were identified, which were classified into 82 GO terms and 14 pathways. Finally, 18 DEGs, which were randomly selected, were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide valuable information for further analysis of the immune defense mechanisms against pathogens in the small intestine of P. sinensis.

Classification and phagocytosis of circulating haemocytes in Chinese mitten crab (Eriocheir sinensis) and the effect of extrinsic stimulation on circulating haemocytes in vivo.

Eriocheir sinensis (Henri Milne Edwards 1854) is one of the most important aquaculture species in China. In this investigation, we characterised the different types of haemocytes of E. sinensis using light and electron microscopy combined with cytochemical analysis and determined the in vivo phagocytic ability of different haemocyte types by injecting polystyrene beads. The haemocytes of E. sinensis were divided into three types: hyalinocytes, semigranulocytes and granulocytes. The hyalinocytes had no or few cytoplasmic granules; the semigranulocytes contained abundant small granules and a few large refractile cytoplasmic granules; and the granulocytes contained numerous large refractile cytoplasmic granules. The hyalinocytes were demonstrated to be the most abundant circulating haemocytes and the most avid phagocytic haemocytes, accounting for approximately 88.7% of the total phagocytes. The haemocyte-containing granules displayed limited phagocytic ability, with approximately 5.0% of granulocytes and 6.3% of semigranulocytes displaying positive phagocytic ability against the invading polystyrene beads in vivo. After injection with Aeromonas hydrophila, Bacillus subtilis and different concentrations of lipopolysaccharide for 0.25, 0.5, 1, 2, 4, 6 and 8 h, all three types of haemocytes experienced dramatic decline and then rapid recovery to their initial levels. A high concentration of lipopolysaccharide and A. hydrophila were extremely toxic to the crabs, as they induced a more serious loss of haemocytes compared with a low concentration of lipopolysaccharide and B. subtilis. Overall, the results obtained in this study indicate that a small proportion of the haemocytes of E. sinensis contributed to the phagocytic process, and the migration of haemocytes and haemocyte lysis were most likely a prominent pathway for pathogen elimination.

Inhibitory effect of acarbose on tyrosinase: application of molecular dynamics integrating inhibition kinetics.

Due to its clinical and cosmetic applications, investigators have paid attention to tyrosinase (TYR) inhibitor development. In this study, a TYR inhibition study with acarbose was investigated to gain insights into the regulation of the catalytic function. Biochemical assay results indicated that acarbose was turned to be an inhibitor of TYR in a reversible binding manner and probed as a distinctive mixed-type inhibitor via measurement of double-reciprocal kinetic (Ki = 18.70 ± 4.12 mM). Time-interval kinetic measurement indicated that TYR catalytic function was gradually inactivated by acarbose in a time-dependent behavior displaying with a monophase process that was evaluated by semi-logarithmic plotting. Spectrofluorimetric measurement by integrating with a hydrophobic residue detector (1-anilinonaphthalene-8-sulfonate) showed that the high dose of acarbose derived a conspicuous local structural deformation of the TYR catalytic site pocket. Computational docking simulation showed that acarbose bound to key residues such as HIS61, TYR65, ASN81, HIS244, and HIS259. Our study extends an understanding of the functional application of acarbose and proposes that acarbose is an alternative candidate drug for a whitening agent via direct retardation of TYR catalytic function and it would be applicable for the relevant skin hyperpigmentation disorders concerning the dermatologic clinical purpose.Communicated by Ramaswamy H. Sarma.

Application of Computational Simulation Integrating Inhibition Kinetics for Detecting Tyrosinase Inhibitor: Salsalate Is a New Inhibitor.

BACKGROUND: Tyrosinase inhibitor developments have been widely attended by investigators for their various applications. OBJECTIVE: A combination of virtual screening of docking simulations and biochemical inhibition kinetics was performed to find a new inhibitor of tyrosinase for the clinical application of an antipigment agent. METHODS: We conducted docking simulations to detect tyrosinase key binding residues and used the detected binding residues to screen the NCBI PubChem database for probing tyrosinase binding compounds. The serial inhibition kinetics and spectrofluorimetry measurements were performed to validate the inhibitory effect on tyrosinase. RESULTS: We have detected 200 candidates and categorized them into four clusters. Among them, we successfully confirmed salsalate as a new inhibitor of tyrosinase measured by serial enzyme kinetics. Salsalate was detected as a reversible inhibitor of tyrosinase displaying a typical mixedtype inhibition manner (IC50 = 22.19 ± 1.01 mM; Ki = 19.98 ± 2.11 mM). Spectrofluorimetry measurement by integrating with 1-anilinonaphthalene-8-sulfonate showed that salsalate mainly induced a slight regional conformation distortion of the tyrosinase active site accompanied by a slight hydrophobic disruption. CONCLUSION: Our study suggests that salsalate is a potential anti-pigment drug via inhibition of tyrosinase activity and it might be applicable for dermatologic clinical application. Also, our study enlarges an insight into the salsalate drug application.

Characterization of the Pelodiscus sinensis polymeric immunoglobulin receptor (P. sinensis pIgR) and its response to LPS and Aeromonas sobria.

The polymeric immunoglobulin receptor (pIgR) is one of the most vital components of mucosal immunity that plays a pivotal role in mediating transcytosis of polymeric immunoglobulin (pIg) on epithelial surfaces for protection against invading pathogens. Herein, we cloned the full-length cDNA of Pelodiscus sinensis pIgR, designated as P. sinensis pIgR, made of an open reading frame (ORF) of 1848 bp, molecular weight of 68.2 kDa and estimated isoelectric point of 7.00. The deduced P. sinensis pIgR sequence had a leader peptide, extracellular region containing four immunoglobulin-like domains (Ig like domains), transmembrane and intracellular regions comparable with other vertebrates. P. sinensis pIgR contained four Ig like domains that corresponded with mammalian D1, D3, D4 and D5 similar with reptile and avian Ig like domains. It had 40 potential phosphorylation sites, four putative N-glycosylation sites and several motifs resembling mammalian pIgR motifs. Phylogenetic analysis showed a close relationship between P. sinensis pIgR with avian and reptile pIgRs. P. sinensis pIgR basal levels were higher in the esophagus, small intestine and intestinnum crissum than in other organs of health turtles. Intragastric delivery of LPS and Aeromonassobria led to significant upregulation of P. sinensis pIgR in tissues of the gastrointestinal tract. A polyclonal anti- P. sinensis pIgR antibody produced in rabbit reacted with the recombinant P. sinensis pIgR protein expressed in Escherichia coli in Western blot. These studies demonstrate the existence and immune response of P. sinensis pIgR to stimulation in mucosal organs in Chinese soft-shelled turtles.

Immunization with Bovine Serum Albumin (BSA) in Oil-Adjuvant Elicits IgM Antibody Response in Chinese Soft-Shelled Turtle (Pelodiscus Sinensis).

Immunoassays are among the frontline methods used for disease diagnosis and surveillance. Despite this, there are no immunoassays developed for the Chinese soft-shelled turtle (Pelodiscus sinensis), which has expanded into large scale commercial production in several Asian countries. One of the critical factors delaying the development of immunoassays is the lack of characterized soft-shelled turtle immunoglobulins. Herein, we used mass spectrometry together with the ProtQuest software to identify the soft-shelled turtle IgM heavy chain in serum, which again was used to produce a polyclonal anti-turtle-IgM in rabbits. Thereafter, the polyclonal anti-turtle-IgM was used as a secondary antibody in an indirect ELISA to evaluate antibody responses of soft-shelled turtles injected with the bovine serum albumin (BSA) model antigen. Our findings show that only turtle immunized with a water-in-oil BSA plus ISA 763A VG adjuvant (SEPPIC, France) emulsion had antibodies detected at 42 days post vaccination (dpv) while turtles injected with phosphate buffered saline (PBS) only as well as turtle injected with BSA dissolved in PBS had no significant antibody levels detected in serum throughout the study period. In summary, our findings show that rabbit polyclonal anti-turtle-IgM produced can be used in ELISA to measure serum antibody responses in immunized soft-shelled turtles. Future studies should explore its application in other immunoassays needed for the disease diagnosis and vaccine development for soft-shelled turtles.

Immune response of peroxinectin of Chinese mitten crab Eriocheir sinensis to exterior stimulation.

Peroxinectin possesses the features of both peroxidase activity and adhesive property and plays important roles in innate immune system of crustaceans. In this study, the sequence of peroxinectin of Eriocheir sinensis (EsPX) was analyzed and its expression in response to exterior stimulation was detected in both in vivo and in vitro examination. We showed that the full-length cDNA sequence was composed of 2701 bp and owned a molecular mass of 85.2 kDa and a theoretical pI (isoelectric point) of 6.91. Real-time PCR revealed that the EsPX was mainly distributed in the muscle, hemocytes and stomach. Furthermore, the EsPX was verified to be located in hyalinocytes, semigranulocytes and granulocytes, and was distributed throughout the cytoplasm and nucleus, especial in cytoplasm. After injected with beads, lipopolysaccharide (LPS) and Aeromonas hydrophila, the EsPX mRNA expression was significantly up-regulated and peaked up at 4, 2 and 16 h respectively (P <0.05). In the in vitro experiment, the stimulation of LPS and beads also induced a prominent boost of EsPX protein in primary cultured hemocytes. The expression of EsPX was peaked up at 4 and 8 h for LPS and beads challenged groups respectively, followed by remarkable release of the incremental EsPX into the extracellular matrix. These findings suggested that the expression of EsPX was susceptible to exterior stimulation, and that the highly expressional EsPX would be released into extracellular matrix by the exterior stimulus.

Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis.

In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 µg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.