A galectin from Eriocheir sinensis functions as pattern recognition receptor enhancing microbe agglutination and haemocytes encapsulation.

Galectins are a family of ß-galactoside binding lectins that function as pattern recognition receptors (PRRs) in innate immune system of both vertebrates and invertebrates. The cDNA of Chinese mitten crab Eriocheir sinensis galectin (designated as EsGal) was cloned via rapid amplification of cDNA ends (RACE) technique based on expressed sequence tags (ESTs) analysis. The full-length cDNA of EsGal was 999 bp. Its open reading frame encoded a polypeptide of 218 amino acids containing a GLECT/Gal-bind_lectin domain and a proline/glycine rich low complexity region. The deduced amino acid sequence and domain organization of EsGal were highly similar to those of crustacean galectins. The mRNA transcripts of EsGal were found to be constitutively expressed in a wide range of tissues and mainly in hepatopancreas, gill and haemocytes. The mRNA expression level of EsGal increased rapidly and significantly after crabs were stimulated by different microbes. The recombinant EsGal (rEsGal) could bind various pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan (GLU), and exhibited strong activity to agglutinate Escherichia coli, Vibrio anguillarum, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and Pichia pastoris, and such agglutinating activity could be inhibited by both d-galactose and α-lactose. The in vitro encapsulation assay revealed that rEsGal could enhance the encapsulation of haemocytes towards agarose beads. These results collectively suggested that EsGal played crucial roles in the immune recognition and elimination of pathogens and contributed to the innate immune response against various microbes in crabs.

Molecular cloning and characterization of a cytoplasmic manganese superoxide dismutase and a mitochondrial manganese superoxide dismutase from Chinese mitten crab Eriocheir sinensis.

Superoxide dismutase (SOD) functions as the first and essential enzyme in the antioxidant system and is ubiquitously existed in both prokaryotes and eukaryotes. In the present study, both cytoplasmic and mitochondrial manganese SOD were identified from Chinese mitten crab Eriocheir sinensis (designed as EscytMnSOD and EsmtMnSOD). The complete nucleotide sequence of EscytMnSOD comprised 1349 bp and consisted of a 5' untranslated regions (UTR) of 43 bp, a 3' UTR of 445 bp and an open reading frame (ORF) of 861 bp encoding a polypeptide of 286 amino acid residues. The full-length cDNA sequence of EsmtMnSOD comprised 990 bp, containing a 5' UTR of 55 bp, a 3' UTR of 278 bp and an ORF of 657 bp encoding a polypeptide of 218 amino acid residues. The deduced amino acid sequences of EscytMnSOD and EsmtMnSOD contained highly conserved MnSOD signature and typical functional domain, and exhibited high similarity with their reported homologues. In the phylogenetic tree, EscytMnSOD and EsmtMnSOD were clustered with their homologues from the land crab Cardisoma armatum. The EscytMnSOD and EsmtMnSOD transcripts were constitutively expressed in haemocytes, muscle, heart, gill, haepatopancreas and gonad, with the highest expression level in gills and haepatopancreas, respectively. The mRNA expression levels of them were all up-regulated in haemocytes with similar profiles after the stimulation of Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The EsmtMnSOD with low basal expression level responded to invading microbes intensely, while the EscytMnSOD with high basal expression level exhibited mild responses against stimulating microbes. The purified rEscytMnSOD and rEsmtMnSOD proteins exhibited specific Mn(2+)-dependent enzymatic activities, while rEscytMnSOD with lower basic activity displayed higher stability than rEsmtMnSOD. All these results indicated that EscytMnSOD and EsmtMnSOD were efficiently antioxidant enzymes and potentially involved in the innate immune responses of E. sinensis with different roles, the former might play a routine role in the innate immune system in crabs, while the later might be involved in the immune response against invading microbes specifically.

Identification and characterization of a serine protease inhibitor Esserpin from the Chinese mitten crab Eriocheir sinensis.

Serine protease inhibitors (serpins) represent an expanding superfamily of endogenous inhibitors that regulate proteolytic events and involve in a variety of physiological processes. A serine protease inhibitor, namely Esserpin, was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) analysis. The full-length cDNA of Esserpin was of 2367 bp, including an open reading frame (ORF) of 1371 bp encoding a polypeptide of 456 amino acids with estimated molecular mass of 49.95 kDa and theoretical isoelectric point of 6.03. A putative signal peptide of 23 amino acids and a classical serpin domain were identified in Esserpin. The deduced amino acid sequence of Esserpin shared homology with serpins from Fenneropenaeus chinensis and Pacifastacus leniusculus. The mRNA transcripts of Esserpin could be detected in all the examined tissues including heart, gill, hemocytes, muscle, gonad and hepatopancreas, and the highest expression level was present in gonad. After the crabs were challenged by Vibrio anguillarum and Pichia pastoris, the expression levels of Esserpin transcripts in hemocytes were significantly up-regulated, and peaked at 24 h (5.18-fold of blank group, P < 0.05) and 3 h (2.87-fold of blank group, P < 0.05), respectively. The functional activity of Esserpin was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombinant Esserpin (rEsserpin) could inhibit trypsin activities in a dose-dependent manner, and it could lead to 100% inhibition of trypsin activities under the concentration of 873.76 nM, while there was no evident inhibition of chymotrypsin observed with rEsserpin. Moreover, rEsserpin inhibited the growth of E. coli at the final concentration of 1747.52 nM, and it also significantly depressed (P < 0.05) the phenoloxidase activity in the plasma at the final concentration of 873.76 nM. These results indicated that Esserpin was a homologue of serpin in crab and it could be induced after immune stimulation and mediate immune response possibly via the inhibition of bacterial growth and the regulation of prophenoloxidase-activating system.

The involvement of PDGF/VEGF related factor in regulation of immune and neuroendocrine in Chinese mitten crab Eriocheir sinensis.

Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in cell proliferation, cell differentiation, and cell migration, vascular development, angiogenesis and neural development. In the present study, a novel PDGF/VEGF related factor gene was cloned and identified in Chinese mitten crab Eriocheir sinensis (designated as EsPVF1). The full-length cDNA of EsPVF1 was of 1173 bp, consisting a 5' untranslated region (UTR) of 54 bp, a 3' UTR of 1131 bp with a poly (A) tail, and an open reading frame (ORF) of 588 bp encoding 196 amino acid residues. A signal peptide of 20 amino acid residues, a PDGF/VEGF homology growth factor domain of 81 amino acids, and a typical cysteine knot motif (CXCXC) were identified in the deduced amino acid sequence of EsPVF1. By fluorescent quantitative real-time PCR, the EsPVF1 mRNA was detected ubiquitously in the select tissues of hemocytes, gonad, heart, muscle, hepatopancreas and gill, with the high abundance in hemocytes and gonad. The mRNA expression level of EsPVF1 was up-regulated and reached the highest at 24 h after Vibrio anguillarum challenge, while it was induced at 3 h, 6 h, 12 h, 24 h and 48 h compared with the untreated group after Pichia pastoris GS115 challenge. Tissue injury also induced the mRNA expression of EsPVF1 in hemocytes of crabs, and the expression level increased obviously at 8 h. The cDNA fragment encoding mature peptide of EsPVF1 was recombined and expressed in Escherichia coli BL21 (DE3) pLysS. Biogenic amine in hemolymph pre-incubated with recombinant protein of EsPVF1 (rEsPVF1) was detected by fluorimetric method. Norepinephrine and dopamine in hemolymph incubated with rEsPVF1 were higher than that in the blank group. Therefore, EsPVF1 could significantly provoke the release of norepinephrine and dopamine. The results collectively indicated that EsPVF1 was involved in regulation of the immune response and neuroendocrine system in crabs.

An interleukin-2 enhancer binding factor 2 homolog involved in immune response from Chinese mitten crab Eriocheir sinensis.

As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates IL-2 gene at level of transcription, splicing and translation in vertebrates and plays significant roles in immune system. In this study, an ILF2 homolog was identified from Chinese mitten crab Eriocheir sinensis (designated as EsILF) by expressed sequence tag (EST) analysis. The full-length cDNA of EsILF was of 2159bp, containing a 5' untranslated region (UTR) of 90bp, a 3' UTR of 866bp with a poly (A) tail, and an open reading frame (ORF) of 1203bp encoding a polypeptide of 400 amino acids with the predicted molecular weight of 44.3kDa, which shared 59.6-64.5% identities with vertebrate ILF2. There were a conserved N-terminal RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain in the primary structure, strongly suggesting that EsILF was a homolog of vertebrate ILF2. The mRNA of EsILF was constitutively expressed in all tested tissues of untreated crabs, including hepatopancreas, gill, gonad, muscle, heart and hemocytes, with highest expression in muscle and relative lower levels in hemocytes and gonad. The mRNA expression of EsILF in hemocytes was regulated differently after the crabs were stimulated by bacteria Listonella anguillarum and fungi Pichia pastoris GS115. The expression level was significantly (P<0.05) down-regulated to 0.35- and 0.29-fold compared with blank group at 6h and 12h after the stimulation of L. anguillarum, while P. pastoris significantly (P<0.05) up-regulated the expression level to 3.2-fold compared with the blank group at 6h post treatment. The results indicated that EsILF was involved in the immune response of crab toward both L. anguillarum and P. pastoris.

A novel type III crustin (CrusEs2) identified from Chinese mitten crab Eriocheir sinensis.

Antimicrobial peptides are important effectors in the host innate immune response against microbial invasion. In the present study, the cDNA encoding a crustin (designated CrusEs2) was cloned from Chinese mitten crab Eriocheir sinensis by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CrusEs2 was of 1237 bp, containing a 5' untranslated region (UTR) of 12 bp, a 3' UTR of 886 bp with a poly (A) tail, and an open reading frame (ORF) of 339 bp encoding a polypeptide of 112 amino acids with a signal peptide of 19 amino acids. The CrusEs2 contained a typical WAP domain, but lacked the Gly-rich domain of the type II crustin and the Cys-rich region present in both type I and type II crustin, suggesting that CrusEs2 should be classified as a type III crustin. The mRNA transcripts of CrusEs2 could be detected in haemocytes and gill, and its expression level in haemocytes was up-regulated after Listonella anguillarum challenge, while decreased after Micrococcus luteus challenge. The mature peptide coding region of CrusEs2 was cloned into pET-21a+ and expressed in Escherichia coli. The purified recombinant CrusEs2 inhibited the growth of Gram-positive bacteria at MIC of 0.093-0.37 µM. The results indicated that CrusEs2 was involved in immune response of E. sinensis against bacterial challenge.

Two novel secreted ferritins involved in immune defense of Chinese mitten crab Eriocheir sinensis.

As a principal extracellular iron storage molecule, secreted ferritin plays an important role in the iron-withholding strategy of innate immunity. In this study, two novel secreted ferritins were identified from Chinese mitten crab Eriocheir sinensis (designated as EsFer-1 and EsFer-2) by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNAs of EsFer-1 and EsFer-2 were of 1278 and 1595 bp, respectively, both containing a putative iron response element (IRE) in their 5' UTRs and multiple A + U-destabilizing elements (TATT or ATTTA) in their 3' UTRs. The ORFs of these two crab ferritin cDNAs were of 639 and 663 bp, respectively, encoding two peptides of 212 and 220 amino acid residues each with a signal peptide and typical structures of ferritins such as four long alpha-helices, one short alpha-helix and an L-loop. EsFer-2 exhibited higher similarity with the H-ferritins from both invertebrates and vertebrates, while EsFer-1 was closer matched to L-ferritins. The eight amino acid residues identified as metal binding sites in vertebrate H-ferritins were conserved in EsFer-2 (Glu53, Tyr60, Glu87, Glu88, His91, Glu146, Glu177 and Gln178), but none of them was observed in EsFer-1. By fluorescent quantitative real-time PCR, mRNA transcripts of EsFer-1 and EsFer-2 were mainly detected in muscle, hepatopancreas and gill, and also marginally detectable in gonad, heart and hemocytes. After the crabs were challenged by bacteria Listonella anguillarum, the transcriptional levels of both EsFer-1 and EsFer-2 in hemocytes were up-regulated twice. In the first up-regulation, the mRNA relative expression levels of both EsFer-1 and EsFer-2 reached peak at 3 h post-challenge, while in the second up-regulation, they did not reach the highest point within the experiment duration. After the fungi Pichia pastoris GS115 challenge, there was only one transcriptional level peak of both the two ferritins, appearing at 6 h post-challenge. These results suggest that secreted EsFer-1 and EsFer-2 are crucial proteins in the iron-withholding defense system, and play important roles in the innate immune responses in crabs.

A putative endocrine factor SIBD (single insulin binding domain protein) involved in immune response of Chinese mitten crab Eriocheir sinensis.

Insulin growth factor binding proteins (IGFBPs), characterized by the conserved insulin binding (IB) domains, are important components of endocrine system and play key roles in metabolism and growth. In the present study, the full-length cDNA of a single IB domain protein (designated EsSIBD) was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) sequence. The 1187 bp EsSIBD cDNA contained a 321 bp open reading frame (ORF) encoding a putative protein of 106 amino acids, a 5'-untranslated region (UTR) of 189 bp, and a 3'-UTR of 677 bp. Multiple sequence alignment presented ten conserved cysteine residues critical for the fundamental structure and function of IB domain. BLAST analysis revealed that EsSIBD shared high similarity with previously known IB domains of IGFBPs with the identities ranging from 40% to 46%. The sequence similarity and domain conservation indicated that EsSIBD was a potential member of the IGFBP family. Phylogenic analysis presented that EsSIBD was closer to IGFBP7 than to the other IB domain containing proteins, suggesting its functional similarity with the endocrine factor IFGBP7. The mRNA expression of EsSIBD in different tissues including hepatopancreas, gill, gonad, muscle, heart and haemocytes, and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EsSIBD mRNA transcripts could be detected in all examined tissues with the highest expression level in gill. The EsSIBD mRNA expression in haemocytes was sensitive to L. anguillarum stimulation and it was up-regulated from 3 to 24 h after challenge. The structure conservation and functional similarity to IFGBPs, and its sensitivity to L. anguillarum stimulation collectively implied that EsSIBD was probably involved in endocrine and immune systems of Chinese mitten crab, and provided insight into the cross-talk between the invertebrate endocrine and immune system.

Erratum: High-Throughput Small Molecule Screen Identifies Modulators of Mitochondrial Function in Neurons.

[This corrects the article DOI: 10.1016/j.isci.2020.100931.].

High-Throughput Small Molecule Screen Identifies Modulators of Mitochondrial Function in Neurons.

We developed a high-throughput assay for modulators of mitochondrial function in neurons measuring inner mitochondrial membrane potential (ΔΨm) and ATP production. The assay was used to screen a library of small molecules, which led to the identification of structural/functional classes of mitochondrial modulators such as local anesthetics, isoflavones, COXII inhibitors, adrenergic receptor blockers, and neurotransmitter system effectors. Our results show that some of the isolated compounds promote mitochondrial health, enhance oxygen consumption rate, and protect neurons against toxic insults found in the cellular environment of Alzheimer disease. These studies offer a set of compounds that may provide efficacy in protecting the mitochondrial system in neurodegenerative disorders.

A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis: cDNA characterization and mRNA expression.

Clip domain serine protease (cSP), characterized by conserved clip domains, is a new serine protease family identified mainly in arthropod, and plays important roles in development and immunity. In the present study, the full-length cDNA of a cSP (designated EscSP) was cloned from Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1380 bp EscSP cDNA contained a 1152 bp open reading frame (ORF) encoding a putative cSP of 383 amino acids, a 5'-untranslated region (UTR) of 54 bp, and a 3'-UTR of 174 bp. Multiple sequence alignment presented twelve conserved cysteine residues and a canonical catalytic triad (His(185), Asp(235) and Ser(332)) critical for the fundamental structure and function of EscSP. Two types of cSP domains, the clip domain and tryp_spc domain, were identified in the deduced amino acids sequence of EscSP. The conservation characteristics and similarities with previously known cSPs indicated that EscSP was a member of the large cSP family. The mRNA expression of EscSP in different tissues and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EscSP mRNA transcripts could be detected in all examined tissues, and were higher expressed in muscle than that in hepatopancreas, gill, gonad, haemocytes and heart. The EscSP mRNA expression in haemocytes was up-regulated after L. anguillarum challenge and peaked at 2 h (4.96 fold, P < 0.05) and 12 h (9.90 fold, P < 0.05). Its expression pattern was similar to prophenoloxidase (EsproPO), one of the components of crab proPO system found in our previous report. These results implied that EscSP was involved in the processes of host-pathogen interaction probably as one of the proPO system members.

Molecular cloning and characterization of peroxiredoxin 6 from Chinese mitten crab Eriocheir sinensis.

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). In this study, the full-length cDNA encoding peroxiredoxin 6 (designated EsPrx6) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5' untranslated region (UTR) of 69 bp, a 3' UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 in various tissues and its temporal expression in haemocytes of crabs challenged with Listonella anguillarum. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E. sinensis.

A thioredoxin with antioxidant activity identified from Eriocheir sinensis.

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 5' untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepatopancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge.

The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab Eriocheir sinensis.

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis.

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8 h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174 bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G+) and Gram-negative (G-) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture.

Drosophila SLC22A Transporter Is a Memory Suppressor Gene that Influences Cholinergic Neurotransmission to the Mushroom Bodies.

The mechanisms that constrain memory formation are of special interest because they provide insights into the brain's memory management systems and potential avenues for correcting cognitive disorders. RNAi knockdown in the Drosophila mushroom body neurons (MBn) of a newly discovered memory suppressor gene, Solute Carrier DmSLC22A, a member of the organic cation transporter family, enhances olfactory memory expression, while overexpression inhibits it. The protein localizes to the dendrites of the MBn, surrounding the presynaptic terminals of cholinergic afferent fibers from projection neurons (Pn). Cell-based expression assays show that this plasma membrane protein transports cholinergic compounds with the highest affinity among several in vitro substrates. Feeding flies choline or inhibiting acetylcholinesterase in Pn enhances memory, an effect blocked by overexpression of the transporter in the MBn. The data argue that DmSLC22A is a memory suppressor protein that limits memory formation by helping to terminate cholinergic neurotransmission at the Pn:MBn synapse.

Identification of genes that promote or inhibit olfactory memory formation in Drosophila.

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/ß and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.

A double WAP domain-containing protein from Chinese mitten crab Eriocheir sinensis with antimicrobial activities against Gram-negative bacteria and yeast.

The whey acidic protein (WAP) domain is characterized by a 'four-disulfide-core' (4-DSC) motif comprising of approximately 50 amino acids with eight highly conserved cysteine residues. Previous research indicated that WAP domain-containing proteins played an important role in the innate immunity of crustaceans. In the present study, a novel double WAP domain (DWD)-containing protein gene was identified from Chinese mitten crab Eriocheir sinensis (designated EsDWD) by expressed sequence tag (EST) analysis and PCR techniques. The full-length cDNA of EsDWD was of 593 bp, consisting of a 5'-terminal untranslated region (UTR) of 71 bp, a 3' UTR of 120 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 402 bp. The ORF encoded a polypeptide of 133 amino acids with the predicted molecular weight of 14.4 kDa and the theoretical isoelectric point of 8.14, including a signal peptide of 22 amino acids and two WAP domains. The EsDWD mRNA transcripts were ubiquitously expressed in all the tested tissues, and its expression level in gill was significantly higher than that in other tissues. The mRNA expression of EsDWD in haemocytes was up-regulated after challenge of Vibrio anguillarum and Pichia pastoris GS115, as well as injury treatment. The cDNA encoding the mature EsDWD protein was cloned and expressed in Escherichia coli BL21 (DE3) pLysS, and the purified recombinant EsDWD (rEsDWD) protein exhibited antimicrobial activities against Gram-negative bacteria V. anguillarum, yeast P. pastoris GS115 and Candida parapsilosis. The results collectively suggested that EsDWD was a novel member of double WAP domain (DWD)-containing proteins, and involved in the immune defense against microorganism and wound healing in E. sinensis.

The involvement of suppressors of cytokine signaling 2 (SOCS2) in immune defense responses of Chinese mitten crab Eriocheir sinensis.

The suppressors of cytokine signaling 2 (SOCS2) has been identified as negative feedback inhibitors for various cytokines signaling via the JAK/STAT pathway. In the present studies, the cDNA of Eriocheir sinensis SOCS2 (designated as EsSOCS2) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsSOCS2 was of 2535bp, consisting of an open reading frame (a ORF) of 1071bp encoding a polypeptide of 357 amino acids. The deduced amino acid sequence of EsSOCS2 shared 55-62% similarity with other SOCS2 family members. There were three typical conserved SOCS family domains in EsSOCS2, including an N-terminal ESS formed from a single amphipathic helix, a central SH2 domain with a classic phosphotyrosine (pY) site and a C-terminal SOCS box. The sequence and structural similarity of EsSOCS2 with SOCS2 proteins from other organisms indicated that EsSOCS2 should be a new member of the SOCS2 family. Phylogenetic analysis revealed that EsSOCS2 was clustered with SOCS2 from the other invertebrates, and fell into the group of type II SOCS subfamily as a sister branch to CIS and SOCS2 from vertebrate, suggesting the great divergence of SOCS2 of vertebrate from invertebrate and complex evolution of SOCS2 family members. The mRNA transcript of EsSOCS2 could be detected by semi-quantitative RT-PCR in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad. The mRNA expression of EsSOCS2 in haemocytes was up-regulated to 3.5-fold at 8h after Listonella anguillarum challenge, 3-fold and 3.5-fold at 4 and 6h, respectively, after Micrococcus luteus challenge. These results collectively suggested that EsSOCS2 could be induced by bacteria challenge, and it was involved in the immune defense responses in E. sinensis.

Molecular characterization and expression of a crustin-like gene from Chinese mitten crab, Eriocheir sinensis.

Crustin is a cysteine-rich antibacterial peptide which widely distributes within decapod crustaceans. In the present study, the cDNA encoding crustin-like peptide (designated CrusEs) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag analysis. The full-length cDNA of CrusEs was of 796bp, containing a 5' untranslated region (UTR) of 214bp, a 3' UTR of 267bp with a poly(A) tail, and an open reading frame (ORF) of 315bp encoding a polypeptide of 104 amino acids including a putative signal peptide of 21 amino acids. A WAP domain and the consensus framework existing in class I crustins were both identified in CrusEs, suggesting that CrusEs is a new member of type I crustins. Quantitative real-time RT-PCR was employed to examine the expression of CrusEs, and its mRNA transcript was mainly detected in haemocytes and gill. The CrusEs mRNA transcript in haemocytes was down-regulated after the challenge with Gram-positive bacteria Micrococcus luteus, while Gram-negative bacteria Listonella anguillarum did not induce significant variation of CrusEs mRNA. The mature peptide of CrusEs was cloned into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli, and the recombinant CrusEs inhibited the growth of Gram-positive bacteria with low MIC. The results indicate that CrusEs is a potent antibacterial protein against Gram-positive bacteria infection, and it may play an important role in innate immune response of E. sinensis.