Chunlin Wang

Effects of ocean acidification on immune responses of the Pacific oyster Crassostrea gigas.

Ocean acidification (OA), caused by anthropogenic CO2emissions, has been proposed as one of the greatest threats in marine ecosystems. A growing body of evidence shows that ocean acidification can impact development, survival, growth and physiology of marine calcifiers. In this study, the immune responses of the Pacific oyster Crassostrea gigas were investigated after elevated pCO2 exposure for 28 days. The results demonstrated that OA caused an increase of apoptosis and reactive oxygen species (ROS) production in hemocytes. Moreover, elevated pCO2 had an inhibitory effect on some antioxidant enzyme activities and decreased the GSH level in digestive gland. However, the mRNA expression pattern of several immune related genes varied depending on the exposure time and tissues. After exposure to pCO2 at ∼2000xa0ppm for 28 days, the mRNA expressions of almost all tested genes were significantly suppressed in gills and stimulated in hemocytes. Above all, our study demonstrated that elevated pCO2 have a significant impact on the immune systems of the Pacific oyster, which may constitute as a potential threat to increased susceptibility of bivalves to diseases.

Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO2

Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO2 condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO2 could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO2 probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C. gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels.

Molecular cloning, genomic structure and antimicrobial activity of PtALF7, a unique isoform of anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus

Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, are becoming predominant candidates for potential therapeutic agents of bacterial and viral diseases. In this study, a unique isoform of ALF (PtALF7) was identified from hemocytes cDNA library of the swimming crab Portunus trituberculatus. The PtALF7 cDNA contained an open reading frame (ORF) of 372 bp encoding 123 amino acids. The deduced peptide of PtALF7 shared high similarity with our previously reported PtALF1-3 but low with PtALF4-6. The PtALF7 gene consisted of three exons interrupted by two introns, and was clearly transcribed from different genomic loci compared with other PtALF isoforms. Totally 128 SNPs including 12 in coding region and 116 in noncoding region were detected in PtALF7 gene by direct sequencing of 20 samples. The mRNA expression of PtALF7 transcript was primarily observed in hemocytes followed by gill and eyestalk, but barely detectable in hepatopancreas. After challenge with Vibrio alginolyticus, a main pathogen causing high mortality in P. trituberculatus, the PtALF7 transcript in hemocytes showed a clear time-dependent response expression pattern with obvious decrease at 6 h and significant increase at 24 h. The recombinant PtALF7 protein exhibited antimicrobial activity against the test Gram-negative and Gram-positive bacteria, but did not inhibit the growth of fungus Pichia pastoris. These results together indicate a potential involvement for PtALF7 in the innate immune response of P. trituberculatus.

Cloning and expression analysis of a transformer gene in Daphnia pulex during different reproduction stages.

The full-length cDNA of a transformer gene (Dptra) was cloned from the cladoceran Daphnia pulex using RACE. Dptra expression was assessed by qPCR and whole-mount in situ hybridization in different reproductive stages. The Dptra cDNA, 1652bp in length, has a 1158-bp open reading frame that encodes a 385 amino acid polypeptide containing one Sex determination protein N terminal (SDP_N) superfamily, eight putative phosphorylation sites, and an arginine-serine (RS)-rich domain at the N-terminus. Dptra showed 81%, 53%, 51% and 45% identity to orthologous genes in Daphnia magna, Apis mellifera, Apis cerana and Bombus terrestris, respectively. Phylogenetic analysis based on deduced amino acid sequences revealed that Dptra clustered in the hymenopteran clade and was most closely related to D. magna and A. mellifera. qPCR showed that Dptra expression increased significantly (P<0.05) in different reproductive stages in the following order: male, ephippial female, parthenogenetic female, resting egg and juvenile female. Dptra expression is significantly different between males and females and it is significantly greater in ephippial females and males than in parthenogenetic D. pulex (with summer eggs). Whole-mount in situ hybridization revealed that Dptra was expressed at different levels between males and females. In males, hybridization signals were found in the first antennae, second antennae and thoracic limb, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. This suggests that the Dptra gene plays significant roles in switching modes of reproduction and in sexual differentiation.

cDNA cloning and mRNA expression of four glutathione S-transferase (GST) genes from Mytilus galloprovincialis

Glutathione S-transferases (GSTs) are phase II enzymes involved in the regulation of redox homeostasis and innate immune responses against bacterial infection, which also play important roles in the detoxification of xenobiotics. In this study, we reported four genes of the GST family (named MgGSTα, MgGSTS1, MgGSTS2, and MgGSTS3, respectively) from Mytilus galloprovincialis. MgGSTα, MgGSTS1, MgGSTS2, and MgGSTS3 consisted of open reading frame (ORF) of 648 bp, 612 bp, 621 bp and 609 bp respectively, which encoded proteins of 216, 204, 207 and 203 amino acids residues, respectively. Sequence analysis showed that the predicted protein sequence of MgGSTs contained the conserved domain of the GST_N and GST_C. Alignment analysis indicated that the MgGSTs were divided into two types, one was of alpha GST, and the others were of sigma class. Tissue distribution study revealed that MgGSTα, MgGSTS2, MgGSTS3 transcripts were highly expressed in hemocytes, while MgGSTS1 mRNA was most abundantly expressed in hepatopancreas. After bacterial challenge, the expression level of these MgGSTs in hemocytes were all significantly higher than that of the control group. These results suggested that MgGSTs might play important roles in the modulation of immune response in M. galloprovincialis.

Gene Transcriptional and Metabolic Profile Changes in Mimetic Aging Mice Induced by D-Galactose

D-galactose injection has been shown to induce many changes in mice that represent accelerated aging. This mouse model has been widely used for pharmacological studies of anti-aging agents. The underlying mechanism of D-galactose induced aging remains unclear, however, it appears to relate to glucose and 1ipid metabolic disorders. Currently, there has yet to be a study that focuses on investigating gene expression changes in D-galactose aging mice. In this study, integrated analysis of gas chromatography/mass spectrometry-based metabonomics and gene expression profiles was used to investigate the changes in transcriptional and metabolic profiles in mimetic aging mice injected with D-galactose. Our findings demonstrated that 48 mRNAs were differentially expressed between control and D-galactose mice, and 51 potential biomarkers were identified at the metabolic level. The effects of D-galactose on aging could be attributed to glucose and 1ipid metabolic disorders, oxidative damage, accumulation of advanced glycation end products (AGEs), reduction in abnormal substance elimination, cell apoptosis, and insulin resistance.

A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn(2+)-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection.

A small heat shock protein (sHSP) from Sinonovacula constricta against heavy metals stresses

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones and involved into many physiological and stress processes. In the present study, the full-length cDNA of sHSP was cloned from razor clam Sinonovacula constricta (denoted as ScsHSP) through cDNA library and PCR approaches. Some feature motifs like the typical α-crystalline domain with six beta strands, three susceptible phosphorylation serines (S(15), S(78), and S(82)) were conserved in the deduce amino acid of ScsHSP. Tissue distribution analysis of the ScsHSP revealed that the mRNA transcripts of ScsHSP were constitutively expressed in all examined tissues with the highest expressions in the haemocytes. The temporal expression of ScsHSP in gill and haemocytes after PbCl2 and CdCl2 exposure were recorded by qPCR. The suppressed expression patterns were detected in CdCl2 stress at both tissues, and the minimum expression were detected at 36 h with 0.58-fold decrease in haemocytes and 0.30-fold in gill compared to each control group. During the PbCl2 exposure experiment, the expression level of ScsHSP increased significantly with larger amplitude in haemocytes. As time progressed, the mRNA transcripts of ScsHSP recovered almost to the original level at 36 h. All our results indicated that ScsHSP was involved into mediating environmental pollutants exposure and considered to be a promising candidate bio-mark for heavy metals monitoring.

Molecular cloning and characterization of two isoforms of cyclophilin A gene from Venerupis philippinarum.

Cyclophilin A (CypA) is a ubiquitously distributed intracellular protein belonging to the immunophilin family, which is recognized as the cell receptor for the potent immunosuppressive drug cyclosporine A. In the present study, two isoforms of cyclophilin A gene (named as VpCypA1 and VpCypA2) were isolated and characterized from Venerupis philippinarum by RACE approaches. Both VpCypA1 and VpCypA2 possessed all conserved features critical for the fundamental structure and function of CypA, indicating that the two isoforms of cyclophilin A should be new members of CypA family. The expression of VpCypA2 mRNA in haemocytes was significantly up-regulated and the highest expression level was detected at 96 h post-infection with 7.7-fold increase compared with that in the blank group. On the contrary, the relative expression level of VpCypA1 mRNA was down-regulated rapidly at 6 h post-infection and reached 0.4-fold of the control group. They exhibited different expression profile and identical effect of immune modulation, which might suggest the two VpCypA isoforms exert their function in a manner of synergy. These results provide valuable information for further exploring the roles of cyclophilin A in the immune responses of V. philippinarum.

Characterization and functional analysis of serine proteinase and serine proteinase homologue from the swimming crab Portunus trituberculatus

Serine proteases (SPs), with their homologues (SPHs), a family of multifunctional proteins, play a crucial role in innate immune system. In our present study, we made an appropriate correction: serine protease homologue PtcSPH (Li et al., [1]) obtained from the swimming crab Portunus trituberculatus was actually a serine protease and re-designated as PtcSP. Sequence analysis revealed PtcSP and PtSP (Li et al., [2]) might be encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Eight exons were identified in genomic DNA sequence of PtcSP. A comprehensive phylogenetic analysis was made combined with our previous reports (Cui et al., [3]; Li et al., [1,2]). The result showed SPs and SPHs of P. trituberculatus had different origins in gene evolution. To further characterize the function(s) of proteins, the recombinant serine proteases or homologues were assayed for various biological functions: proteinase activity, antimicrobial activity and microorganisms binding activity. The recombinant protein PtcSP exhibited trypsin-like protease activity and antibacterial activity. PtSPH1 (Li et al., [2]) lacked proteolytic activity but displayed binding activity to yeast and the crab pathogenic bacterium, Vibrio alginolyticus. Further, the N-terminal clip domain of PtcSP had antibacterial activity and the C-terminal SP-like domain had trypsin-like protease activity.