Xiaowei Shi

The modulation of catecholamines to the immune response against bacteria Vibrio anguillarum challenge in scallop Chlamys farreri

Catecholamines are pivotal signal molecules in the neuroendocrine-immune regulatory network, and implicated in the modulation of immune response. In the present study, the activities of some immune-related enzymes and the concentration of catecholamines were determined in circulating haemolymph of scallops Chlamys farreri after bacteria Vibrio anguillarum challenge. The activities of superoxide dismutase (SOD), catalase (CAT) and lysozyme (LYZ) increased significantly and reached 610 U mg(-1) at 12 h, 37.6 U mg(-1) at 6 h and 261.5 U mg(-1) at 6 h after bacteria challenge, respectively. The concentration of norepinephrine, epinephrine and dopamine also increased significantly and reached 114.9 ng mL(-1) at 12 h, 86.9 ng mL(-1) at 24 h and 480.4 pg mL(-1) at 12 h after bacteria challenge, respectively. Meanwhile, the activities of these immune-related enzymes in haemolymph were monitored in those scallops which were challenged by bacteria V. anguillarum and stimulated simultaneously with norepinephrine, epinephrine and adrenoceptor antagonist. The injection of norepinephrine and epinephrine repressed significantly the induction of bacteria challenge on the activities of immune-related enzymes, and they were reduced to about half of that in the control groups. The blocking of α and β-adrenoceptor by antagonist only repressed the increase of CAT and LYZ activities significantly, while no significant effect was observed on the increase of SOD activities. The collective results indicated that scallop catecholaminergic neuroendocrine system could be activated by bacteria challenge to release catecholamines after the immune response had been triggered, and the immune response against bacteria challenge could been negatively modulated by norepinephrine, epinephrine, and adrenoceptor antagonist. This information is helpful to further understand the immunomodulation of catecholamines in scallops.

The phenoloxidase activity and antibacterial function of a tyrosinase from scallop Chlamys farreri

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.

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot(-1), 0.40 ± 0.06 U mgprot(-1), P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot(-1), 93.0 ± 6.3 nmol mgprot(-1), P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.

The Immunomodulation of Acetylcholinesterase in Zhikong Scallop Chlamys farreri

Background Acetycholinesterase (AChE; EC 3.1.1.7) is an essential hydrolytic enzyme in the cholinergic nervous system, which plays an important role during immunomodulation in vertebrates. Though AChEs have been identified in most invertebrates, the knowledge about immunomodulation function of AChE is still quite meagre in invertebrates. Methodology A scallop AChE gene was identified from Chlamys farreri (designed as CfAChE), and its open reading frame encoded a polypeptide of 522 amino acids. A signal peptide, an active site triad, the choline binding site and the peripheral anionic sites (PAS) were identified in CfAChE. The recombinant mature polypeptide of CfAChE (rCfAChE) was expressed in Pichia pastoris GS115, and its activity was 71.3±1.3 U mg−1 to catalyze the hydrolysis of acetylthiocholine iodide. The mRNA transcripts of CfAChE were detected in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in hepatopancreas. The relative expression level of CfAChE mRNA in haemocytes was both up-regulated after LPS (0.5 mg mL−1) and human TNF-α (50 ng mL−1) stimulations, and it reached the highest level at 12 h (10.4-fold, P<0.05) and 1 h (3.2-fold, P<0.05), respectively. After Dichlorvos (DDVP) (50 mg L−1) stimulation, the CfAChE activity in the supernatant of haemolymph decreased significantly from 0.16 U mg−1 at 0 h to 0.03 U mg−1 at 3 h, while the expression level of lysozyme in the haemocytes was up-regulated and reached the highest level at 6 h, which was 3.0-fold (P<0.05) of that in the blank group. Conclusions The results collectively indicated that CfAChE had the acetylcholine-hydrolyzing activity, which was in line with the potential roles of AChE in the neuroimmune system of vertebrates which may help to re-balance the immune system after immune response.

The arginine kinase in Zhikong scallop Chlamys farreri is involved in immunomodulation

Arginine kinase (AK) catalyzes the reversible phosphorylation of l-arginine to form phosphoarginine, and plays a critical role in energy metabolism in invertebrates. In the present study, a scallop AK gene was identified from Chlamys farreri with an open reading frame (ORF) of 1101bp encoding for a protein of 366 amino acids (designed as CfAK). An ATP-gua PtransN domain which was described as a guanidine substrate specificity domain (GS domain) and an ATP-gua Ptrans domian which was responsible for binding ATP, were both identified in CfAK. The mRNA transcripts of CfAK were detectable in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in the muscle and the lowest level in the hemocytes. The expression level of CfAK mRNA increased from fertilized eggs to eyebot, and reached the highest in the trochophore stage. The relative expression level of CfAK mRNA in muscle was up-regulated significantly after LPS (0.5mg/mL) stimulation, and reached the peak at 6h (5.2-fold, P<0.05). The activity of inducible nitric oxide synthase (iNOS) in the supernatant of muscle homogenate increased significantly from 3.2U/mg at 0 h to 9.7 U/mg at 12h after LPS stimulation, while the concentration of nitric oxide (NO) in the supernatant of muscle homogenate began to increase at 3h (21.55 μmol/L), and reached the top concentration at 24h (42.27 μmol/L), then recovered to the normal level after 48 h. The recombinant protein of CfAK (rCfAK) expressed in Escherichia coli displayed Arginine kinase activity, and its apparent K(m) was 0.82 ± 0.11 and 1.24 ± 0.13 mM for L-arginine and ATP-Na, respectively. The results indicated that the CfAK was involved in energy production and utilization during the whole life process, and might refer to the immunomodulation process via altering the NO concentration and iNOS activity in scallop Chlamys farreri.

A Dopa Decarboxylase Modulating the Immune Response of Scallop Chlamys farreri

Background Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate. Methodology The full-length cDNA encoding DDC (designated CfDDC) was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P<0.05) at 3 h and reached the peak at 12 h (9.8-fold, P<0.05), and then recovered to the baseline level. The recombinant protein of CfDDC (rCfDDC) was expressed in Escherichia coli BL21 (DE3)-Transetta, and 1 mg rCfDDC could catalyze the production of 1.651±0.22 ng dopamine within 1 h in vitro. When the haemocytes were incubated with rCfDDC-coated agarose beads, the haemocyte encapsulation to the beads was increased significantly from 70% at 6 h to 93% at 24 h in vitro in comparison with that in the control (23% at 6 h to 25% at 24 h), and the increased haemocyte encapsulation was repressed by the addition of rCfDDC antibody (which is acquired via immunization 6-week old rats with rCfDDC). After the injection of DDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (P<0.05) of blank group at 12 h and 0.47-fold (P<0.05) at 24 h, respectively. Conclusions These results collectively suggested that CfDDC, as a homologue of DDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc.

Acetylcholine modulates the immune response in Zhikong scallop Chlamys farreri.

Acetylcholine (ACh) is an indispensable neurotransmitter and neuromodulator in the cholinergic nervous system and it is implicated in the dynamic modulation of immune response in vertebrates. Although ACh has also been identified in most invertebrates, the knowledge about its immunomodulation is still limited. In the present study, the immunoreactivities of ACh and acetylcholinesterase (AChE) were observed in all the tested tissues of scallop Chlamys farreri, including adductor muscle, mantle, gill, hepatopancreas, kidney and gonad. The ACh concentration in the supernate of scallop hemolymph increased from 11.59 ± 0.27 to 14.36 ± 0.17 μM L(-1) at 6 h after LPS (0.5 mg ml(-1)) stimulation, and increased to 15.51 ± 1.20 μM L(-1) at 3 h after the stimulation of tumor necrosis factor alpha (TNF-α) (50 ng ml(-1)). After LPS stimulation, the mRNA expression levels of superoxide dismutase (CfSOD), catalase (CfCAT) and lysozyme (CfLYZ) in hemocytes increased significantly at 3 h (P < 0.05), 6 h (P < 0.05) and 12 h (P < 0.05), respectively. Compared with the LPS treatment, the induction of CfSOD, CfCAT and CfLYZ expression in hemocytes was repressed effectively (P < 0.05) by the co-stimulation of LPS and ACh (5 × 10(-7) M) at 3 h (P < 0.05), 6 h (P < 0.05) and 12 h (P < 0.05), respectively. Furthermore, the expression level of CfCAT in hemocytes increased significantly after 12 h by the co-stimulation with LPS and ACh (P < 0.05). These results indicated collectively that the scallop cholinergic nervous system could be activated by immune stimulations, and it might play an essential role in immunomodulation of scallops.

A novel cold-regulated cold shock domain containing protein from scallop Chlamys farreri with nucleic acid-binding activity.

Background The cold shock domain (CSD) containing proteins (CSDPs) are one group of the evolutionarily conserved nucleic acid-binding proteins widely distributed in bacteria, plants, animals, and involved in various cellular processes, including adaptation to low temperature, cellular growth, nutrient stress and stationary phase. Methodology The cDNA of a novel CSDP was cloned from Zhikong scallop Chlamys farreri (designated as CfCSP) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full length cDNA of CfCSP was of 1735 bp containing a 927 bp open reading frame which encoded an N-terminal CSD with conserved nucleic acids binding motif and a C-terminal domain with four Arg-Gly-Gly (RGG) repeats. The CSD of CfCSP shared high homology with the CSDs from other CSDPs in vertebrate, invertebrate and bacteria. The mRNA transcripts of CfCSP were mainly detected in the tissue of adductor and also marginally detectable in gill, hepatopancreas, hemocytes, kidney, mantle and gonad of healthy scallop. The relative expression level of CfCSP was up-regulated significantly in adductor and hemocytes at 1 h and 24 h respectively after low temperature treatment (P<0.05). The recombinant CfCSP protein (rCfCSP) could bind ssDNA and in vitro transcribed mRNA, but it could not bind dsDNA. BX04, a cold sensitive Escherichia coli CSP quadruple-deletion mutant, was used to examine the cold adaptation ability of CfCSP. After incubation at 17°C for 120 h, the strain of BX04 containing the vector pINIII showed growth defect and failed to form colonies, while strain containing pINIII-CSPA or pINIII-CfCSP grew vigorously, indicating that CfCSP shared a similar function with E. coli CSPs for the cold adaptation. Conclusions These results suggest that CfCSP is a novel eukaryotic cold-regulated nucleic acid-binding protein and may function as an RNA chaperone in vivo during the cold adaptation process.

The immunomodulation of nicotinic acetylcholine receptor subunits in Zhikong scallop Chlamys farreri.

Nicotinic acetylcholine receptor (nAChR), the best-studied ionotropic neuron receptor protein, is a key player in neuronal communication, and it has been reported to play an important role in immunomodulation of vertebrates. Although nAChRs have also been identified in most invertebrates, the knowledge about their immunomodulation is still limited. In the present study, two scallop nAChR genes were identified from Chlamys farreri (designed as CfnAChR1 and CfnAChR2), which encoded 384 and 443 amino acids, respectively. The conserved disulfide-linked cystines, ion selectivity residues and the hydrophobic gating residues (L251, V255 and V259) were identified in CfnAChR1 and CfnAChR2. The immunoreactivities of CfnAChR1 and CfnAChR2 were observed in all the tested scallop tissues, including adductor muscle, mantle, gill, hepatopancreas, kidney and gonad. After LPS (0.5 mg mL(-1)) stimulation, the expression of CfnAChR1 mRNA in haemocytes increased significantly by 9.83-fold (P < 0.05) and 12.93-fold (P < 0.05) at 3 h and 24 h, respectively. While the expression level of CfnAChR2 mRNA increased 43.94% at 12 h after LPS stimulation (P < 0.05). After TNF-α (50 ng mL(-1)) stimulation, the expression levels of CfnAChR1 and CfnAChR2 both increased significantly at 1 h, which were 21.33-fold (P < 0.05) and 2.44-fold (P < 0.05) of that in the PBS group, respectively. The results collectively indicated that the cholinergic nervous system in scallops could be activated by immune stimulations through CfnAChR1 and CfnAChR2, which function as the links between the cholinergic nervous system and immune system.

The molecular characterization of a catalase from Chinese mitten crab Eriocheir sinensis.

Catalase (CAT) is an antioxidant enzyme and plays a significant role in the protection against oxidative stress by reducing hydrogen peroxide. The CAT cDNA of Eriocheir sinensis (EsCAT) was cloned via RACE technique. The complete sequence of EsCAT cDNA consisted of a 5′ untranslated regions (UTR) of 224 bp, a 3′ UTR of 1287 bp with a poly (A) tail and an open reading frame (ORF) of 1542 bp, which encoded a polypeptide of 513 amino acid residues with a calculated molecular mass of approximately 58.86 kDa and a theoretical isoelectric point of 6.880. The deduced amino acid sequence of EsCAT contained a highly conserved proximal active‐site signature motif (60FDRERIPERVVHAKGAL76) and a proximal heme–ligand signature motif (350RLFSYNDTH358) and exhibited high similarity with other reported CATs. In the phylogenetic tree, EsCAT was clustered with the CATs from Scylla serrata and Portunus trituberculatus. The EsCAT transcripts were constitutively expressed in haepatopancreas, haemocytes, gill, gonad, muscle and heart, with highest expression level in haepatopancreas. The relative expression level of EsCAT mRNA in haemocytes was continuously up‐regulated and reached the peak level at 48 h post‐Vibrio anguillarum challenge. The purified recombinant EsCAT protein displayed antioxidant activity against hydrogen peroxide with high thermal stability and broad spectrum of pH values. All these results demonstrated that EsCAT was an efficient antioxidant enzyme and potentially involved in the regulation of redox and innate immune response of crabs.