题目: An Mpeg (Macrophage expressed gene) from the Pacific oyster Crassostrea gigas: molecular characterization and gene expression 

作者: He, X., Y. Zhang, Z. Yu*

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,30:870-876. 

摘要: Mpegs (macrophage expressed genes) encode members of the MACPF (membrane-attack complex/perforin) protein superfamily that play essential roles in innate immunity. In the present study, a homolog of Mpeg1 was identified in Crassostrea gigas and designed Cg-Mpeg1. The complete cDNA of Cg-Mpeg1 is 2781 bp in length, containing an ORF of 2226 bp, which encodes a putative protein of 742 amino acids with a predicted 19-aa hydrophobic signal peptide, an MACPF domain, and a transmembrane domain. Phylogenetic analysis shows that Cg-Mpeg1 is similar to other mollusk MACPF proteins and might originate in an ancient ancestor gene before the divergence of protostomes and deuterostomes. Localization study revealed that Cg-Mpeg1 protein is found primarily in late endosomes. The MACPF domain from Cg-Mpeg1 exhibits significant antibacterial activity to both Gram-negative and positive bacteria. Furthermore, Real-time Quantitative PCR analysis showed that Cg-Mpeg1 is expressed in all tissues detected with highest expression in gill and gonads. Moreover, Mpeg1 mRNA levels are significantly up-regulated following infection with Vibrio alginolyticus. These results highlight that Cg-Mpeg1 plays an essential role in host defense and elimination of pathogens in C. gigas. 

题目: The second bactericidal permeability increasing protein (BPI) and its revelation of the gene duplication in the Pacific oyster, Crassostrea gigas 

作者: Zhang, Y., X. He, X. Li, D. Fu, Chen, J., Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,30:954-963. 

摘要: A novel homolog of BPI was cloned from the hemocyte cDNA of Crassostrea gigas and designed as Cg-BPI2, which share the highest sequence identity with the well-known Cg-BPI (designed as Cg-BPI1). The complete cDNA of Cg-BPI2 included an open reading frame (ORF) of 1440 bp, and 3′ and 5′ untranslated regions (UTR’s) of 49 bp and 166 bp, respectively. The ORF encoded a putative protein of 479 amino acids with predicted 22-aa hydrophobic signal peptide. The phylogenetic analysis showed that one of the gene duplications could have resulted in the emergence of two homologs of BPI in oysters, which probably might have occurred after the gastropod-bivalve divergence. Furthermore, molecular modeling analysis showed that both Cg-BPIs are similar to a highly extended boomerang like shape of human BPI, consisting of an N- and C-terminal barrel and a central β-sheet. Comparison of the electrostatic surface potentials revealed that surfaces of Cg-BPI2 have more intense positive charge than that of human BPI and the Cg-BPI1. The recombinant N-terminal barrel domain showed a high affinity to LPS and can effectively kill Gram-negative bacteria. The mRNAs of two Cg-BPIs were observed in all tissues examined with the highest expression in gills. The mRNAs expression profiles in response to bacterial challenge revealed that they were inducible under infection, but with a distinct and complementary expression patterns between Cg-BPI1 and Cg-BPI2. Our findings of this second BPI gene demonstrated presence of its gene duplication for the first time in invertebrate and it appears to be one of effective LPS-binding AMPs in elimination of Gram-negative pathogens C. gigas. 

题目: Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge. 

作者: Fu, D., J. Chen, Y. Zhang, Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011, 31: 118-125. 

摘要: Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3′, 5′-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion. 

题目: F-type lectin involved in defense against bacterial infection in the pearl oyster (Pinctada martensii). 

作者: Chen, J., Xiao S., Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,30: 750-754. 

摘要: In invertebrates and vertebrates, carbohydrate-binding proteins (lectins) play an important role in innate immunity against microbial invasion. In the present study, we report the cloning of an F-type lectin (designated as PmF-lectin) from pearl oyster (Pinctada martensii) using a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of PmF-lectin contains an open reading frame (ORF) of 579 bp coding for192 amino acids. The deduced polypeptide possesses six conserved residues of the F-lectin family critical for the formation of disulfide bonds (Cys43-Cys143, Cys75-Cys76 and Cys102-Cys119). Reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR) analyses in adult tissues showed that the PmF-lectin mRNA was abundantly expressed in haemocytes and gill, moderately expressed in the mantle, and rarely expressed in other tissues tested. After challenge with Vibrio alginolyticus, expression of PmF-lectin mRNA in haemocytes was dramatically up-regulated, reaching the highest level (13-fold higher than that of the control group) at 3 h post challenge, and then dropped gradually. These results suggest that PmF-lectin is a member of the F-lectin family and is involved in the innate immune response in pearl oyster. 

题目: The first homolog of a TRAF7 (TNF receptor-associated factor 7) gene in a mollusk, Crassostrea hongkongensis. 

作者: Fu, D., Y. Zhang, S. Xiao and Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,31: 1208-1210 

摘要: Tumor necrosis factor (TNF) receptor-associated factor 7 (TRAF7) is one of several adaptor proteins that are critically involved in the activation of TLR-dependent NF-κB signaling. In this report, the first mollusk TRAF7 (designated ChTRAF7) homolog was isolated from Crassostrea hongkongensis by screening a suppression subtractive library. The full-length cDNA, 2290 bp in length, encodes a putative protein of 686 amino acids that contains a RING finger domain, an adjacent zinc finger domain, and seven WD40 repeats. ChTRAF7 is ubiquitously expressed in various tissues including digestive gland, mantle, gill, heart, hemocytes, muscle, and gonads, with the highest expression observed in gonads. Temporal expression of ChTRAF7 following bacterial infection shows that expression of ChTRAF7 in hemocytes decreases from 2 to 12 h post-challenge, and then recovered to the original level after 24 h. These results indicate that ChTRAF7 may play an important role in signal transduction in the immune response of oysters. 

题目: Cloning of the microsomal glutathione S-transferase 3 (MGST3) gene and expression analysis in pearl oyster Pinctada martensii. 

作者: Chen J, Xiao S, Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,31:  823-830. 

摘要: crosomal glutathione S-transferase (MGST) functions in cellular defense against xenobiotics and provides protection against the action of lipid hydroperoxides produced as a consequence of oxidative stress. In this study, a full-length cDNA encoding MGST3 (referred to as PmMGST3) was identified from the pearl oyster,Pinctada martensii by a combination of expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmMGST3 is 971 bp and contains a 5′ UTR of 39 bp, a 3′ UTR of 491 bp with a canonical polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) of 447 bp encoding a polypeptide of 146 residues. The deduced polypeptide contains a conserved motif (FNCx1QRx2H) characteristic of the MGST3 subfamily. The PmMGST3 transcript could be detected in all tissues tested, with highest transcript level seen in hepatopancreas. Cadmium treatment significantly increased PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depressed mRNA levels and then increased its level in haemocytes, gill and hepatopancreas in a time-dependent manner. In an assay using cumene hydroperoxide as a substrate, we demonstrated that PmMGST3 possesses glutathione-dependent peroxidase activity. These results suggest that PmMGST3 plays an important role in cellular defense against oxidative stress caused by cadmium and bacteria. 

题目: Cloning of the microsomal glutathione S-transferase 3 (MGST3) gene and expression analysis in pearl oyster Pinctada martensii. 

作者: Chen J, Xiao S, Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,31:  823-830. 

摘要: crosomal glutathione S-transferase (MGST) functions in cellular defense against xenobiotics and provides protection against the action of lipid hydroperoxides produced as a consequence of oxidative stress. In this study, a full-length cDNA encoding MGST3 (referred to as PmMGST3) was identified from the pearl oyster,Pinctada martensii by a combination of expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmMGST3 is 971 bp and contains a 5′ UTR of 39 bp, a 3′ UTR of 491 bp with a canonical polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) of 447 bp encoding a polypeptide of 146 residues. The deduced polypeptide contains a conserved motif (FNCx1QRx2H) characteristic of the MGST3 subfamily. The PmMGST3 transcript could be detected in all tissues tested, with highest transcript level seen in hepatopancreas. Cadmium treatment significantly increased PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depressed mRNA levels and then increased its level in haemocytes, gill and hepatopancreas in a time-dependent manner. In an assay using cumene hydroperoxide as a substrate, we demonstrated that PmMGST3 possesses glutathione-dependent peroxidase activity. These results suggest that PmMGST3 plays an important role in cellular defense against oxidative stress caused by cadmium and bacteria. 

题目: Molecular cloning, characterization and expression analysis of receptor for activated C kinase 1 (RACK1) from pearl oyster (Pinctada martensii) challenged with bacteria and exposed to cadmium. 

作者: Chen, J., Xiao S., Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011, 31: 781-787. 

摘要: Receptor for activated C kinase 1 (RACK1) is involved in superoxide anion generation and play an important role in the immune response. In the study, we cloned the full-length sequence of pearl oyster, Pinctada martensii, RACK1 (designated as PmRACK1) by a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmRACK1 is 1176 bp in length, containing a 5′ UTR of 83 bp, a 3′ UTR of 139, and an open reading frame (ORF) of 954 bp encoding 317 amino acids. Analysis of protein domain features showed that the deduced polypeptide contain seven WD domains characteristic of RACK1 protein family. The tissue distribution of PmRACK1 in unchallenged pearl oysters and temporal expression pattern of PmRACK1 in pearl oysters challenged with bacteria and exposed to 0.1 ppm cadmium were analyzed by quantitative real-time PCR (qRT-PCR). The transcript was detected in all tissues tested, and the expression level was highest in hepatopancreas and lowest in adductor muscle. After challenge with bacteria, expression level of PmRACK1 in haemocytes was gradually decreased until 6 h post challenge, and then up-regulated over time. After exposure to cadmium, its expression level in gill decreased on 1 d post exposure, and then increased as time elapsed, and its expression level in hepatopancreas gradually decreased until 2 d post exposure, and then increased over time. These results suggested that PmRACK1 was involved in oxidative stress response caused by bacteria and cadmium and was a useful biomarker for cadmium exposure. The expression pattern of PmRACK1 in response to bacterial challenge also has a potential link with organism’s immune response. 

题目: A novel sialic acid binding lectin from Hong Kong oyster (Crassostrea hongkongensis) with anti-bacterial activity.

作者: He, X., Y. Zhang, and Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,31:1247-1250 

摘要: Lectins play an important role in immune recognition and host defense. In the present study, a full-length cDNA encoding a novel sialic acid binding lectin was cloned from Crassostrea hongkongensis (designatedCh-salectin) by rapid amplification of cDNA ends (RACE). It is 531 bp in length, containing a 21 bp 5′ UTR, a 39 bp 3′ UTR and a 468 bp ORF coding for 156 amino acids. The Ch-salectin protein contains a signal peptide and a conserved complement component C1q domain. The purified recombinant MBP-tagged Ch-salectin protein can bind to a sialic acid containing protein fetuin and significantly inhibit the growth of both Gram-negative and Gram-positive bacteria. Furthermore, the transcription of Ch-salectin was inducible and significantly up-regulated during Vibrio alginolyticus infection. Thus, these results highlight the essential roles of Ch-salectin in immune recognition and host defense against bacterial infection in C. hongkongensis. 

题目: Two homologues of catalase genes are involved in host protection against bacterial infection and oxidative stress in Crassostrea hongkongensis. 

作者: Zhang, Y., D. Fu, F. Yu, Q. Liu, Z. Yu* 

刊物: Fish Shellfish Immunol.，（IF=3.044，I区）

刊号: 2011,31:894-903. 

摘要: Catalase is one of the key antioxidant enzymes and it appears to be involved in protection against immune infection and oxidative stress. Here, two catalase cDNAs (ChCat-1 and ChCat-2) were isolated from hemocytes of Crassostrea hongkongensis using SSH and RACE. The full-length cDNAs of ChCat-1 andChCat-2 are 1913 and 2466 bp in length, encoding proteins of 515 and 511 amino acids, respectively. Multiple alignments of amino acid sequences revealed that both ChCat-1 and ChCat-2 possess several characteristic features of the catalase family of enzymes, including one proximal active site signature, one heme-ligand signature, and three catalytic amino acid residues (His72, Asn145 and Tyr355). Phylogenetic analysis indicates that these two catalases may share a common ancestral gene and result from a gene duplication event following the divergence of bivalves and gastropods. Constitutive expression of ChCat-1and ChCat-2 was observed in all tissues studied, with highest levels of expression in gill and muscle, respectively. The expression of both genes was inducible by bacterial infection, and reached the maximum at 8 h (9.0-fold) and 12 h (2.3-fold) post-infection, respectively. Furthermore, both the purified ChCat-1 and ChCat-2 protein displayed a strong catalase activity, and S2 cells carrying ChCat-1 or ChCat-2 showed a higher degree of resistance to H2O2 than that of control cells. In a word, this is the first report of the presence of two catalase genes in a single marine bivalve, and our results highlight the involvement of both ChCat-1 and ChCat-2 in host protection against pathogen infection and oxidative stress in C. hongkongensis.