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1.
Mol Immunol ; 139: 50-64, 2021 11.
Article En | MEDLINE | ID: mdl-34454185

To unveil the neuroendocrine-immune (NEI) mechanism of crustaceans under high ambient ammonia-N, crustacean hyperglycemic hormone (CHH) in L. vannamei was knocked down under 20 mg/L ammonia-N exposure. The results showed that the expression of CHH in the eyestalks decreased significantly when CHH was silenced. After CHH was knocked down, the levels of CHH, ACh, DA, NE, and 5-HT in the haemolymph decreased significantly. Correspondingly, the expressions of GC, ACh7R, DM1, DA1R, and 5-HT7R in haemocytes down-regulated significantly, while DA4R and α2AR up-regulated significantly. Besides, the expression of Toll3 reduced significantly. And significantly changes occurred in the levels of G protein effectors (AC and PLC), second messengers (cAMP, cGMP, CaM, and DAG), protein kinases (PKA, PKC and PKG), and nuclear transcription factors (CREB, Dorsal, Relish and NKRF). Furthermore, immune defense proteins (BGBP and PPO3, Crustin A, ALF, LYC, TNFα, and IL-16), phagocytosis-related proteins (Cubilin, Integrin, Peroxinectin, Mas-like protein, and Dynamin-1) and exocytosis-related proteins (SNAP-25, VAMP-2 and Syntaxin) changed significantly. Eventually, a significant decrease in the levels of THC, haemocytes phagocytosis rate, plasma PO, antibacterial and bacteriolytic activities was detected. Therefore, these results indicate that under ammonia-N stress, the combination of CHH and GC mainly affects exocytosis of shrimp through the cGMP-PKG-CREB pathway. Simultaneously, CHH stimulates the release of biogenic amines, and then activate G protein effectors after binding to their specific receptors, to regulate exocytosis mainly via the cAMP-PKA-CREB pathway and influence phagocytosis primarily by the cAMP-PKA-NF-κB pathway. CHH can enhance ACh, and then activate G protein effectors after binding to the receptors, and finally regulate exocytosis mainly through the cAMP-PKA-CREB pathway and regulate phagocytosis by the cAMP-PKA-NF-κB pathway. CHH can also promote Toll3-NF-κB pathway, thereby affecting the expressions of immune defense factors. This study contributes to a further understanding of the NEI mechanism of crustacean in response to environmental stress.


Arthropod Proteins/immunology , Immunity, Innate/immunology , Invertebrate Hormones/immunology , Nerve Tissue Proteins/immunology , Penaeidae/immunology , Stress, Physiological/immunology , Ammonia/toxicity , Animals , Arthropod Proteins/metabolism , Environment , Invertebrate Hormones/metabolism , Nerve Tissue Proteins/metabolism , Penaeidae/metabolism , Signal Transduction/immunology
2.
Fish Shellfish Immunol ; 93: 559-566, 2019 Oct.
Article En | MEDLINE | ID: mdl-31330256

Shrimps like other arthropods rely on innate immune system, and may have some form of adaptive immunity in defending against pathogens. Phagocytosis is one of the oldest cellular processes, serving as a development process, a feeding mechanism and especially as a key defense reaction in innate immunity of all multicellular organisms. It is confirmed that crustacean hyperglycemic hormone (CHH) is one of the most important neuropeptides produced by Neuro-endocrine Immune (NEI) regulatory network, which undertakes important roles in various biological processes, especially in immune function and stress response. In this study, the recombinant Litopenaeus vannamei CHH (rLvCHH) was obtained from a bacterial expression system and the intracellular signaling pathways involved in the mechanism of phagocytosis after rLvCHH injection was investigated. The results showed that the contents of adenylyl cyclase (AC), phospholipase C (PLC) and calmodulin (CaM) in hemocytes were increased significantly after rLvCHH injection. Furthermore, the mRNA expression levels of NF-kB family members (relish and dorsal) and phagocytosis-related proteins in hemocytes were basically overexpressed after rLvCHH stimulation, while the expression level of NF-kB repressing factor (NKRF) gene was down-regulated significantly. Eventually, the total hemocyte count and phagocytic activity of hemocyte were dramatically enhanced within 3 h. Collectively, these results indicate that shrimps L. vannamei could carry out a simple but 'smart' NEI regulation through the action of neuroendocrine factors, which could couple with their receptors and trigger the downstream signaling pathways during the phagocytic responses of hemocytes.


Arthropod Proteins/immunology , Hemocytes/immunology , Immunity, Innate/genetics , Invertebrate Hormones/immunology , Nerve Tissue Proteins/immunology , Penaeidae/genetics , Penaeidae/immunology , Phagocytosis/immunology , Animals , Arthropod Proteins/genetics , Dose-Response Relationship, Drug , Invertebrate Hormones/genetics , Nerve Tissue Proteins/genetics , Random Allocation , Signal Transduction/immunology
3.
Fish Shellfish Immunol ; 72: 544-551, 2018 Jan.
Article En | MEDLINE | ID: mdl-29158205

The molting-inhibiting hormones (MIHs) from the crustacean hyperglycemic hormone (CHH) family are a group of neuropeptides that are implicated in regulation of molting and reproduction in crustaceans. In this study, a novel protein containing a typical crustacean neuropeptide domain was identified from Litopenaeus vannamei. The protein showed high homology with other shrimp MIHs and was then designated as a MIH-like protein (MIHL). Among the detected tissues, the heart expressed the highest level of MIHL. The expression of MIHL could be significantly up-regulated after infection with white spot syndrome virus (WSSV), gram-negative bacterium Vibro parahaemolyticus and gram-positive bacterium Staphylococcus aureus, indicating that MIHL could be involved in immune responses. The promoter of MIHL was predicted to contain two NF-κB binding sites and could be regulated by the NF-κB family protein Relish but not Dorsal, suggesting that MIHL could be an effector gene of the IMD/Relish pathway. Silencing of MIHL in vivo by RNAi strategy significantly down-regulated the expression of many immune effector genes and increased the mortalities of shrimp infected by V. parahaemolyticus and WSSV and their copy numbers in tissues. These confirmed that MIHL could play a role in antiviral and antibacterial immune responses in shrimp.


Gene Expression Regulation/immunology , Immunity, Innate/genetics , Invertebrate Hormones/genetics , Invertebrate Hormones/immunology , Neuropeptides/genetics , Neuropeptides/immunology , Penaeidae/genetics , Penaeidae/immunology , Amino Acid Sequence , Animals , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Arthropod Proteins/immunology , Base Sequence , Gene Expression Profiling , Invertebrate Hormones/chemistry , Neuropeptides/chemistry , Phylogeny , Sequence Alignment , Staphylococcus aureus/physiology , Vibrio parahaemolyticus/physiology , White spot syndrome virus 1/physiology
4.
Gen Comp Endocrinol ; 233: 32-42, 2016 07 01.
Article En | MEDLINE | ID: mdl-27179884

Moulting in crustaceans is regulated by moult-inhibiting hormone (MIH) of the CHH family neuropeptides. The inhibitory functions of MIH have pivotal roles in growth and reproduction of Penaeus monodon. In this study, we report the expression of a thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231bp, that codes for 77 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The translation expression vector construct (mf-PmMIH I+pET32a+) upon induction produced 29.85kDa mature MIH I fusion protein (mf-PmMIH I). The purified fusion protein was used as exogenous MIH I and as antigen to raise polyclonal antisera. When fusion protein (mf-PmMIH I) was injected into D2 and D3 stages of juvenile shrimp, the moult cycle duration was extended significantly to 16.67±1.03 and 14.67±1.03days respectively compared to that of 11.67±1.03days in controls. Moult duration was further reduced to 8.33±0.82days when polyclonal antiserum (anti-mf-PmMIH I - 1:500 dilutions) was injected. Anti-mf-PmMIH I immunolocalized MIH I producing neurosecretory cells in the eyestalk of P. monodon. In short, the present manuscript reports an innovative means of moult regulation in P. monodon with thioredoxin fused MIH I and antisera developed.


Antibodies/pharmacology , Invertebrate Hormones/pharmacology , Molting/drug effects , Penaeidae , Recombinant Fusion Proteins/pharmacology , Thioredoxins/pharmacology , Animals , Antibodies/immunology , Female , Immune Sera/pharmacology , Invertebrate Hormones/genetics , Invertebrate Hormones/immunology , Invertebrate Hormones/metabolism , Mice , Mice, Inbred BALB C , Molting/physiology , Penaeidae/drug effects , Penaeidae/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Thioredoxins/genetics , Thioredoxins/immunology , Thioredoxins/metabolism , Time Factors
5.
Biol Reprod ; 90(1): 12, 2014 Jan.
Article En | MEDLINE | ID: mdl-24337313

Levels of vitellogenin (VG) and vitellogenesis-inhibiting hormone (VIH) in the whiteleg shrimp, Litopenaeus vannamei, were measured by time-resolved fluoroimmunoassay in relation to the molting cycle and ovarian maturation induced by eyestalk ablation. During the molt cycle, VG mRNA expression levels and VG concentrations showed similar patterns of fluctuation. VG levels increased significantly at early intermolt (stage C0) in adults, but not in subadults. Unilateral and bilateral eyestalk ablation increased VG levels in adults, whereas only bilateral eyestalk ablation affected subadults. VIH levels showed contrasting patterns between adults and subadults. In adults, levels were high in late postmolt adults (stage B) and then low thereafter, whereas they increased from postmolt (stage A) to intermolt (stage C0) in subadults and remained high. Unilateral eyestalk ablation increased VIH levels 10 days following ablation in adults, after which levels decreased at 20 days. VIH levels decreased from 10 to 20 days after bilateral ablation. Both unilateral and bilateral ablation led to increased VIH levels in subadults. Eyestalk ablation induced ovarian maturation, but did not reduce VIH concentrations in the hemolymph. This phenomenon was perhaps due to other crustacean hyperglycemic hormone peptides having cross-reactivity with VIH antibodies. This is the first report to quantify concentrations of VG and VIH together in L. vannamei hemolymph, and to examine their relative dynamics.


Carrier Proteins/genetics , Invertebrate Hormones/genetics , Molting/physiology , Penaeidae , Vitellogenins/genetics , Animals , Antibodies/metabolism , Carrier Proteins/immunology , Carrier Proteins/metabolism , Eye Enucleation , Female , Gene Expression Regulation, Developmental , Hemolymph/metabolism , Invertebrate Hormones/immunology , Invertebrate Hormones/metabolism , Penaeidae/genetics , Penaeidae/growth & development , Vitellogenins/isolation & purification , Vitellogenins/metabolism
6.
PLoS One ; 7(3): e34510, 2012.
Article En | MEDLINE | ID: mdl-22470576

BACKGROUND: Bursicon is a heterodimer neuropeptide composed of two cystine knot proteins, bursicon α (burs α) and bursicon ß (burs ß), that elicits cuticle tanning (melanization and sclerotization) through the Drosophila leucine-rich repeats-containing G protein-coupled receptor 2 (DLGR2). Recent studies show that both bursicon subunits also form homodimers. However, biological functions of the homodimers have remained unknown until now. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we show in Drosophila melanogaster that both bursicon homodimers induced expression of genes encoding antimicrobial peptides (AMPs) in neck-ligated adults following recombinant homodimer injection and in larvae fat body after incubation with recombinant homodimers. These AMP genes were also up-regulated in 24 h old unligated flies (when the endogenous bursicon level is low) after injection of recombinant homodimers. Up-regulation of AMP genes by the homodimers was accompanied by reduced bacterial populations in fly assay preparations. The induction of AMP expression is via activation of the NF-κB transcription factor Relish in the immune deficiency (Imd) pathway. The influence of bursicon homodimers on immune function does not appear to act through the heterodimer receptor DLGR2, i.e. novel receptors exist for the homodimers. CONCLUSIONS/SIGNIFICANCE: Our results reveal a mechanism of CNS-regulated prophylactic innate immunity during molting via induced expression of genes encoding AMPs and genes of the Turandot family. Turandot genes are also up-regulated by a broader range of extreme insults. From these data we infer that CNS-generated bursicon homodimers mediate innate prophylactic immunity to both stress and infection during the vulnerable molting cycle.


Drosophila Proteins/metabolism , Drosophila melanogaster/immunology , Gene Expression Regulation , Invertebrate Hormones/metabolism , Transcription Factors/metabolism , Animals , Antimicrobial Cationic Peptides/metabolism , Dimerization , Drosophila Proteins/genetics , Drosophila Proteins/immunology , Drosophila melanogaster/growth & development , Immunity, Innate , Invertebrate Hormones/immunology , Larva , Molting/genetics , Transcription Factors/genetics
7.
Microb Pathog ; 50(3-4): 179-91, 2011.
Article En | MEDLINE | ID: mdl-21195157

Recently the cDNA coding for anti-lipopolysaccharide factor (ALF) has been identified from the Indian mud crab, Scylla serrata and has been named S. serrata anti-lipopolysaccharide factor (SsALF). SsALF protein sequence demonstrated the presence of two highly conserved cystine residues between which the putative lipopolysaccharide (LPS) binding domain is known to be located. In this study, we have designed and synthesized a 24 amino acid linear (lSsALF24) and a cyclic (cSsALF24) peptides based on this putative LPS binding domain and demonstrated the ability of these peptides to bind to LPS. The peptides were active against vaginal pathogens demonstrated by MIC, CFU and phagocytosis assays. cSsALF24 did not show toxicity to human vaginal epithelial cells (HeLa-S3), macrophages and rabbit erythrocytes even at high concentration (64.64 µM). Flow cytometry results demonstrated that cSsALF24 peptide suppressed LPS induced phagocytosis of FITC labeled E. coli. HeLa cells were stimulated with LPS (10 µg/ml) alone for 6 h or after two washings with PBS, treated for 1 h with cSsALF24 (64.64 µM). After washing, the cells were cultured for 24 h in fresh media. The spent media as well as cells were collected for the determination of cytokine/chemokine levels such as interleukin-6 (IL-6) interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and interleukin-1α (IL-1α) using ELISA and RT-PCR respectively. Similar results were obtained with LPS stimulated cells treated with c/nSsALF24 or unstimulated cells treated with c/nSsALF24. The expression of cytokine/chemokines and mRNA's coding these proteins were unaffected in c/nSsALF24 treated cells. In contrast, in LPS stimulated cells, the expression levels of these molecules were up-regulated via the induction of nuclear factor kappa-B (NF-kB) levels. However, the expression of these pro-inflammatory markers was decreased in LPS stimulated cells following the treatment with cSsALF24, attributing anti-inflammatory potential of the peptide. Collectively, these findings suggest that cSsALF24 might regulate the vaginal epithelial cell immune responses indirectly through modulation of LPS-TLR4 binding in NF-kB pathway.


Anti-Bacterial Agents/immunology , Invertebrate Hormones/immunology , Lipopolysaccharides/immunology , NF-kappa B/immunology , Peptides/immunology , Vagina/immunology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Brachyura/immunology , Cell Line , Female , Humans , Invertebrate Hormones/chemistry , Lipopolysaccharides/chemistry , Macrophages/drug effects , Macrophages/immunology , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Phagocytosis , Rabbits , Signal Transduction
8.
Fish Shellfish Immunol ; 30(1): 58-66, 2011 Jan.
Article En | MEDLINE | ID: mdl-20883796

Antimicrobial peptides (AMPs) serve a major role in host defense systems against microbial invasion. In this study, a novel isoform (ALFSp2) of antilipopolysaccharide factors (ALFs) was cloned from the mud crab, Scylla paramamosain. The open reading frame of the ALFSp2 cDNA is 348 bp and encodes for a predicted 115 amino acid residues (12.92 kDa), and a mature protein of 94 amino acids and a molecular mass of 10.79 kDa. The amino acid sequence of ALFSp2 has an overall similarity of 74%, 66% and 52% to those of Eriocheir sinensis ALF, Penaeus monodon ALFPm3 and S. paramamosain ALFSp1, respectively. The genomic organization of the ALFSp2 gene consists of three exons and two introns, whilst the upstream region contains multiple putative transcription factor binding sites. In healthy crabs, ALFSp2 transcript levels were high in the hemocytes and gill tissues, intermediate levels in the intestine and muscles and at a low level in the hepatopancreas, as determined by RT-PCR. To characterize the in vitro antimicrobial activities of ALFSp2, the 24 amino acid LPS-binding domain encoding peptide was synthesized and revealed an antimicrobial activity against Gram-positive (Aerococcus viridans and Micrococcus luteus) and Gram-negative (Vibrio harveyi and Vibrio anguillarum) bacteria. Altogether these results suggest a potential involvement for ALFSp2 in the defense mechanism of the mud crab, S. paramamosain.


Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Bacteria/drug effects , Brachyura/metabolism , Genomics , Invertebrate Hormones/genetics , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Base Sequence , Cloning, Molecular , Gills/metabolism , Hemocytes/metabolism , Hepatopancreas/metabolism , Intestinal Mucosa/metabolism , Invertebrate Hormones/immunology , Molecular Sequence Data , Muscles/metabolism , Phylogeny , Protein Isoforms
9.
Fish Shellfish Immunol ; 30(2): 583-91, 2011 Feb.
Article En | MEDLINE | ID: mdl-21168510

Anti-lipopolysaccharide factors (ALFs), as the potent antimicrobial peptides, can bind and neutralize lipopolysaccharide (LPS) and exhibit broad spectrum antimicrobial activities. In this study, three isoforms of the ALF homologues (PtesALF1-3) were identified from eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA sequences of PtesALF1, 2 and 3 were 1138, 1052 and 1057 bp encoding 92, 108 and 123 amino acids, respectively. PtesALF1-3 contained two conserved cysteine residues and shared high similarity with other reported ALFs. Predicted tertiary structures of PtesALF2 and 3 containing four ß-strands and three α-helix were similar to that described in Limulus polyphemus, while PtesALF1 had only one α-helix in its spatial structure. Sequence analysis revealed PtesALF1-3 were encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Totally 89 SNPs including 18 in coding region and 71 in noncoding region were detected by direct sequencing of 30 genomic samples. The mRNA expression of PtesALF1 and PtesALF1-3 transcripts was mainly detected in haemocytes but showed different expression pattern in other tissues including hepatopancreas, gill, eyestalk and muscle. After challenge with Vibrio alginolyticus, the temporal expression level of PtesALF1-3 transcripts in haemocytes showed a clear time-dependent response expression pattern with two peaks within the experimental period of 32 h, while PtesALF1 was up-regulated only once with obvious decrease at 6 h and significant increase at 24 h. These results suggest that the PtesALF isoforms have different tissue specificity and might provide multiple protective functions against invading bacteria in P. trituberculatus.


Brachyura/genetics , Brachyura/immunology , Amino Acid Sequence , Animals , Base Sequence , Brachyura/classification , Gene Expression Profiling , Gene Expression Regulation , Gene Library , Invertebrate Hormones/chemistry , Invertebrate Hormones/genetics , Invertebrate Hormones/immunology , Phylogeny , Protein Isoforms , Sequence Alignment , Sequence Homology, Amino Acid
10.
Dev Comp Immunol ; 34(9): 945-52, 2010 Sep.
Article En | MEDLINE | ID: mdl-20416335

The anti-lipopolysaccharide factor (ALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724bp, consisting of an open reading frame (ORF) of 363bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53-65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional alpha-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonella anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris, indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture.


Gram-Negative Bacterial Infections/immunology , Invertebrate Hormones/metabolism , Lipopolysaccharides/metabolism , Listonella/immunology , Mycoses/immunology , Pichia/immunology , Shellfish , Amino Acid Sequence , Animals , Anti-Infective Agents/immunology , Anti-Infective Agents/metabolism , Blood Coagulation , Brachyura , Cloning, Molecular , Gene Expression Profiling , Gram-Negative Bacterial Infections/genetics , Gram-Negative Bacterial Infections/metabolism , Immunity, Innate/genetics , Invertebrate Hormones/genetics , Invertebrate Hormones/immunology , Lipopolysaccharides/antagonists & inhibitors , Listonella/pathogenicity , Molecular Sequence Data , Mycoses/genetics , Mycoses/metabolism , Pichia/pathogenicity , Sequence Homology
11.
Fish Shellfish Immunol ; 28(4): 661-71, 2010 Apr.
Article En | MEDLINE | ID: mdl-20060477

A novel antistasin-like cDNA homologue named as Ab-Antistasin was isolated from the disk abalone Haliotis discus discus normalized cDNA library. The Ab-Antistasin (1398-bp) consisted of an 1185-bp open reading frame encoding 395 amino acid (aa) residues. The predicted molecular mass and isoelectric point of Ab-Antistasin was 44 kDa and 8.5, respectively, and showed highest identity (23.1%) to Hydra magnipapillata antistasin. The most striking feature of Ab-Antistasin is the 12-fold internal repeats (IR) of an antistasin-like domain. Ten of the 12 IR domains (26-27 aa) are highly conserved, with 6 cysteines and 1 glycine. Ab-Antistasin was comprised of three Bowman-Birk serine protease inhibitor family motifs. The recombinant Ab-Antistasin (rAb-Antistasin) was over-expressed in Escherichia coli and purified using a pMAL system. rAb-Antistasin (10 microM) was able to inhibit trypsin activity by 66% in a dose-dependent manner. Moreover, it exhibited low prolongation activity for coagulation in an APTT assay (86.0 s compared to control 42.0 s) with human blood. Endogenous Ab-Antistasin mRNA was found to be expressed in digestive tract, hepatopancreas, hemocytes, abductor muscle and mantle, with highest expression levels in digestive tract followed by hepatopancreas and hemocytes. Quantitative real time PCR results revealed that Ab-Antistasin transcription was significantly induced at 3 h post-infection (p.i.) after challenged by a mixture of bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes) in the abalone digestive tract; in the hemocytes, induction occurred at 6 and 12 h. The results indicated that Ab-Antistasin could play an important role in the immune responses of mollusks.


Bacterial Physiological Phenomena , Gastropoda/immunology , Gastropoda/microbiology , Gene Expression Regulation , Invertebrate Hormones/genetics , Invertebrate Hormones/immunology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Gastropoda/classification , Gastropoda/genetics , Invertebrate Hormones/chemistry , Molecular Sequence Data , Phylogeny , Sequence Alignment , Sequence Homology, Amino Acid
12.
J Gen Virol ; 90(Pt 6): 1491-1498, 2009 Jun.
Article En | MEDLINE | ID: mdl-19264668

The anti-lipopolysaccharide factor (ALF) from the black tiger shrimp, Penaeus monodon, has been shown previously to exhibit a broad spectrum of activity against various strains of bacteria and fungi. Herein, the recombinant ALFPm3 (rALFPm3) protein was examined for its role in the defence against white spot syndrome virus (WSSV) infection in haematopoietic (Hpt) cell cultures of the freshwater crayfish, Pacifastacus leniusculus, as well as in live P. monodon shrimps. Incubation of Hpt cell cultures with a mixture of WSSV and rALFPm3 resulted in a dose-dependent decrease in VP28 gene expression levels, compared with those incubated with WSSV alone, with an rALFPm3 IC50 value lower than 2.5 microM. However, pre-treatment of Hpt cells with 5 microM rALFPm3 showed no induced protection against subsequent WSSV infection, whereas the synthetic crayfish ALF peptide could protect cells at a higher concentration (10 microM). The in vivo role of ALFPm3 was examined by injection of P. monodon with WSSV pre-treated with rALFPm3 protein. The results clearly showed that rALFPm3 was able to reduce WSSV propagation and prolong the survival of shrimps.


Invertebrate Hormones/immunology , Penaeidae/immunology , Penaeidae/virology , Virus Diseases/prevention & control , White spot syndrome virus 1/immunology , Animals , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides , Arthropod Proteins , Astacoidea , Cells, Cultured , Invertebrate Hormones/pharmacology , Survival Analysis , White spot syndrome virus 1/drug effects
13.
Mol Immunol ; 45(7): 1916-25, 2008 Apr.
Article En | MEDLINE | ID: mdl-18078996

Antimicrobial peptides are an essential component of the innate immune system of most organisms. Expressed sequence tag analysis from various shrimp (Litopenaeus vannamei) tissues revealed transcripts corresponding to two distinct sequences (LvALF1 and LvALF2) with strong sequence similarity to anti-lipopolysaccharide factor (ALF), an antimicrobial peptide originally isolated from the horseshoe crab Limulus polyphemus. Full-length clones contained a 528bp transcript with a predicted open reading frame coding for 120 amino acids in LvALF1, and a 623bp transcript with a predicted open reading frame coding for 93 amino acids in LvALF2. A reverse genetic approach was implemented to study the in vivo role of LvALF1 in protecting shrimp from bacterial, fungal and viral infections. Injection of double-stranded RNA (dsRNA) corresponding to the LvALF1 message resulted in a significant reduction of LvALF1 mRNA transcript abundance as determined by qPCR. Following knockdown, shrimp were challenged with low pathogenic doses of Vibrio penaeicida, Fusarium oxysporum or white spot syndrome virus (WSSV) and the resulting mortality curves were compared with controls. A significant increase of mortality in the LvALF1 knockdown shrimp was observed in the V. penaeicida and F. oxysporum infections when compared to controls, showing that this gene has a role in protecting shrimp from both bacterial and fungal infections. In contrast, LvALF1 dsRNA activated the sequence-independent innate anti-viral immune response giving increased protection from WSSV infection.


Antimicrobial Cationic Peptides/immunology , Bacterial Infections/veterinary , Immunity/immunology , Invertebrate Hormones/immunology , Mycoses/veterinary , Penaeidae/immunology , Amino Acid Sequence , Animals , Bacterial Infections/immunology , Biological Assay , Gene Expression Profiling , Gene Silencing/drug effects , Immunity/drug effects , Invertebrate Hormones/chemistry , Invertebrate Hormones/genetics , Invertebrate Hormones/metabolism , Molecular Sequence Data , Mycoses/immunology , Penaeidae/drug effects , Penaeidae/microbiology , Penaeidae/virology , Phylogeny , RNA, Double-Stranded/administration & dosage , RNA, Double-Stranded/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Survival Rate , White spot syndrome virus 1/drug effects
14.
Mol Immunol ; 44(12): 3195-203, 2007 May.
Article En | MEDLINE | ID: mdl-17368541

Antilipopolysaccharide factors (ALFs) are small basic proteins that can bind and neutralize lipopolysaccharide (LPS) and have broad spectrum antimicrobial activities. In this study, we describe the isolation of the full-length cDNA encoding for ALF peptide (ALFSp) of mud crab, Scylla paramamosain by sequencing a hemocyte cDNA library and using the rapid amplification cDNA end (RACE) method. A full-length ALFSp cDNA of 614 bp contains an open reading frame (ORF) of 372 bp, encoding 123 amino acid protein with 26 residues signal sequence. The calculated molecular mass of the mature protein is 11.18 kDa. The highly two conserve cysteine residues and putative LPS binding domain were observed in ALFSp peptide. Comparison of amino acid sequences revealed that ALFSp shared high identity with other known ALFs and had an overall similarity of 65, 64, 63, 61 and 59% to those of Fenneropenaeus chinensis, Litopenaeus vannamei, Marsupenaeus japonicus, Limulus polyphemus, and Tachypleus tridentatus, respectively. A neighbour-joining tree showed a clear differentiation of each species and also indicated that ALF from S. paramamosain, Carcinus maenas and Callinectes sapidus are closely related phylogenetically. The genomic DNA sequence of ALFSp gene consists of 1075 bp containing three exons and two introns. Tissue distribution analysis revealed that ALFSp was abundantly expressed in hemocytes, intestine, and muscle but not in eyestalk. The synthetic ALFSp peptide containing putative LPS binding domain revealed a strong antimicrobial activity against several bacteria especially on the growth of Gram-positive bacteria, Micrococcus luteus and Gram-negative bacteria, Vibrio harveyi suggested that ALFSp could play an essential role in defense mechanism in S. paramamosain.


Arthropods/genetics , Invertebrate Hormones/genetics , Animals , Antimicrobial Cationic Peptides , Arthropod Proteins , Arthropods/immunology , Binding Sites , Cloning, Molecular , Invertebrate Hormones/immunology , Lipopolysaccharides
15.
Mol Immunol ; 43(13): 2061-9, 2006 May.
Article En | MEDLINE | ID: mdl-16442159

LPS is known as an effective stimulator of the immune system in various animals, including mammals and horseshoe crabs (HSC). Both of these animal groups have suppressive regulatory proteins for the LPS response, e.g. the bactericidal/permeability increasing protein in mammals and anti-LPS factor (ALF) in HSC. Prawns are a valuable aquaculture species, but the regulatory molecules and/or mechanisms that respond to LPS are largely unknown. To investigate the molecular mechanism of the LPS response in kuruma prawns, we cloned a cDNA having a LPS binding domain. A full-length cDNA gene, denoted as M-ALF (Marsupenaeus japonicus ALF-like peptide) was cloned that consisted of 746bp and encoded 123 amino-acid residues. The 3' non-translated region of this gene had the pentamer of ATTTA repeated four times; this is known as sequences for messenger RNA stabilization. Deduced amino-acid sequences showed a 42% homology with Japanese HSC-ALF. In particular, both have clusters of basic and hydrophobic amino acids, indicating that the region is probably binding to lipid A. The mRNA expression was determined for hemocytes, lymphoid organs, hearts, intestines and gills by RT-PCR. The mRNA expression was augmented 1.5-3h after LPS administration in lymphoid organs, but then decreased to normal level at 6h. Synthetic peptides containing Cys30 to Cys51 had LPS neutralizing activity to the Limulus reaction and NO production in RAW264.7 cells. These data suggest that in kuruma prawns, M-ALF acts as a LPS regulator during the acute phase response after invasion of pathogens.


Invertebrate Hormones/genetics , Penaeidae/genetics , 3' Untranslated Regions/genetics , 3' Untranslated Regions/immunology , Acute-Phase Reaction/genetics , Acute-Phase Reaction/immunology , Animals , Antimicrobial Cationic Peptides , Arthropod Proteins , Cell Line , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/immunology , Gene Expression Regulation/immunology , Horseshoe Crabs/genetics , Horseshoe Crabs/immunology , Invertebrate Hormones/immunology , Invertebrate Hormones/pharmacology , Lipid A/immunology , Lipid A/pharmacology , Mice , Nitric Oxide/biosynthesis , Nitric Oxide/immunology , Penaeidae/immunology , Protein Binding/genetics , Protein Binding/immunology , RNA Stability/drug effects , RNA Stability/genetics , RNA Stability/immunology , RNA, Messenger/genetics , RNA, Messenger/immunology , Sequence Homology, Amino Acid
16.
Dev Comp Immunol ; 29(10): 841-51, 2005.
Article En | MEDLINE | ID: mdl-15978281

Anti-lipopolysaccharide factors (ALFs), originally characterized from horseshoe crabs, have been recently identified from hemocytes of the black tiger shrimp, Penaeus monodon, by a genomic approach. In order to characterize the properties and biological activities of this immune effector in shrimp, ALFPm3, the most abundant isoform found in P. monodon, was expressed in the yeast Pichia pastoris. Large-scale production in fermentor provided 262 mg/l of recombinant ALFPm3 which was purified to homogeneity by single chromatography step on expanded-bed Streamline SP6XL. The rALFPm3 was further characterized in terms of N-terminal sequencing and mass spectrometry. Anti-microbial assays demonstrated that rALFPm3 has a broad spectrum of anti-fungal properties against filamentous fungi, and anti-bacterial activities against both Gram-positive and Gram-negative bacteria, associated with a bactericidal effect. Interestingly, rALFPm3 is highly efficient against various Vibrio species including strains pathogenic for shrimp. Finally, a synthetic peptide corresponding to a part of the putative LPS-binding site of ALFPm3 was shown to display activities mainly directed against Gram-positive bacteria indicating the involvement of the full molecule to the anti-microbial activity for Gram-negative bacteria.


Anti-Infective Agents/immunology , Invertebrate Hormones/genetics , Lipopolysaccharides/immunology , Penaeidae/genetics , Penaeidae/immunology , Amino Acid Sequence , Animals , Anti-Infective Agents/pharmacology , Base Sequence , DNA, Complementary/genetics , Fungi/drug effects , Gene Expression , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Hemocytes/immunology , Invertebrate Hormones/immunology , Microbial Sensitivity Tests , Molecular Sequence Data , Penaeidae/microbiology , Pichia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , Transformation, Genetic , Vibrio/drug effects , Vibrio/immunology , Vibrio/pathogenicity
17.
Alcohol Alcohol ; 39(5): 386-92, 2004.
Article En | MEDLINE | ID: mdl-15289211

AIMS: To examine the immune consequences of chronic alcoholism in man, in relation to the known association between alcoholism and raised incidence and severity of infections. METHODS: In 36 alcoholics without liver disease, at the point of commencing withdrawal from alcohol, the following measures of immune competence were measured: the immunophenotypes of cells, acute phase proteins, the endotoxin-neutralizing capacity (ENC) of the serum, titers of anti-lipopolysaccharide (LPS) antibodies, and ex vivo cytokine inducibility in T cells and monocytes (TNFalpha, IL1beta, IL1RA, IL4, IL6, IL8, IL10 and IL12). The results were compared to those from healthy volunteers (day controls). Measures were repeated after 8-13 days of abstinence. RESULTS: LPS-binding protein (LBP) and soluble CD14 (sCD14) were significantly increased in patients' sera at the outset of withdrawal, whereas reduced titers of anti-LPS IgG (P = 0.012) and a reduced ENC (P = 0.001) were measured. Only ENC rapidly returned to normal values after withdrawal therapy. Cytokine induction with phorbol ester showed no significant alterations in patients' T cells. Patients' monocytes, however, responded to LPS stimulation with enhanced IL1beta-, but reduced TNFalpha- and IL12-production (P = 0.004, P = 0.0042 and P = 0.001, respectively). While IL1- and TNFalpha-responses normalized after the withdrawal period, impairment of the IL12 response persisted throughout the observation period of 2 weeks. CONCLUSIONS: Alcoholism results in a prolonged LPS-mediated hypoinflammatory conditioning of the innate but not the adaptive immune system, which is not reversed immediately after withdrawal. This alcohol-induced status of the immune system predisposes to infections and sepsis by blunting initial response to the pathogens.


Alcoholism/immunology , Alcoholism/physiopathology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Immunoglobulins/immunology , Interleukins/immunology , Lipopolysaccharide Receptors/immunology , Tumor Necrosis Factor-alpha/immunology , Alcoholism/therapy , Antimicrobial Cationic Peptides , Arthropod Proteins , Carrier Proteins/biosynthesis , Chronic Disease , Enzyme-Linked Immunosorbent Assay , Ethanol/adverse effects , Flow Cytometry , Humans , Immunoglobulins/blood , Invertebrate Hormones/immunology , Lipopolysaccharide Receptors/biosynthesis , Lipopolysaccharide Receptors/blood , Male , Physical Examination , Substance Withdrawal Syndrome/blood , Substance Withdrawal Syndrome/etiology , Surveys and Questionnaires
18.
Peptides ; 23(5): 853-62, 2002 May.
Article En | MEDLINE | ID: mdl-12084515

In crustaceans, the synthesis of ecdysteroid molting hormones is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced by an eyestalk neuroendocrine system, the X-organ/sinus gland complex. Using sequence analysis software, two regions of the blue crab (Callinectes sapidus) MIH peptide were selected for antibody production. Two 14-mer peptides were commercially synthesized and used to generate polyclonal antisera. Western blot analysis revealed that each antiserum bound to proteins of the predicted size in extracts of C. sapidus sinus glands, and lysates of insect cells containing recombinant MIH. Thin section immunocytochemistry using either antiserum showed specific immunoreactivity in X-organ neurosecretory cell bodies, their associated axons and collaterals, and their axon terminals in the sinus gland.


Antibodies/immunology , Decapoda/chemistry , Invertebrate Hormones/analysis , Invertebrate Hormones/immunology , Amino Acid Sequence , Animals , Axons/chemistry , Blotting, Western , Decapoda/anatomy & histology , Decapoda/cytology , Immunohistochemistry , Invertebrate Hormones/chemical synthesis , Invertebrate Hormones/chemistry , Molecular Sequence Data , Organ Specificity , Rabbits , Sequence Alignment , Sequence Homology, Amino Acid
19.
Cell Tissue Res ; 307(2): 243-54, 2002 Feb.
Article En | MEDLINE | ID: mdl-11845331

The crustacean hyperglycaemic hormones (cHHs) are multifunctional neuropeptides that play a central role in the physiology of crustaceans. A partial cDNA coding for cHH of the Norway lobster, Nephrops norvegicus, was cloned; this cDNA was fused to glutathione- S-transferase (GST) to obtain a recombinant fusion protein that was used to raise a rabbit antiserum and to perform a biological assay. The specificity of the purified antibody was demonstrated by means of Western blotting. To validate the specificity of the purified antibody to the cHH of N. norvegicus and its cross-reactivity with other species, we performed standard immunocytochemistry of the eyestalk on: (1) paraffin sections of the decapod species N. norvegicus, Munida rugosa and Astacus leptodactylus and of the stomatopod Squilla mantis; (2) semithin resin sections of N. norvegicus and Palaemon elegans; (3) ultrathin sections of N. norvegicus sinus gland (transmission electron microscopy studies). The pattern of immunoreactivity shown by N. norvegicus eyestalk sections conforms to distribution, relative amount and ultrastructural features of cHH-containing neurons and nerve endings as reported in the previous literature. In all the crustacean species examined, the antibody marks precisely the X organ-sinus gland complex and unspecific staining is completely lacking. In addition, its specific cross-reaction by immunoprecipitation depletes shrimp eyestalk extract of hyperglycaemic activity in an in vivo bioassay. The results obtained show a cHH-specific molecular recognition despite the fact that the species tested belong to systematic groups increasingly remote in the phylogenetic tree. The antibody could be used for advancing our knowledge on cHH activity in a variety of crustacean species, e.g. for monitoring reproductive and stress conditions.


Antibodies/metabolism , Crustacea/metabolism , Endocrine Glands/physiology , Nerve Tissue Proteins/immunology , Animals , Antibody Specificity , Arthropod Proteins , Astacoidea/metabolism , Biological Assay , Cross Reactions , Decapoda/metabolism , Eye/chemistry , Eye/ultrastructure , Glutathione Transferase/genetics , Invertebrate Hormones/immunology , Nephropidae/metabolism , Neurons/ultrastructure , Rabbits/immunology , Recombinant Proteins/immunology , Species Specificity
20.
J Immunol ; 167(5): 2694-9, 2001 Sep 01.
Article En | MEDLINE | ID: mdl-11509613

Among the most frequent anaphylactic reactions to insects are those attributed to reduviid bugs. We report the purification and identification of the major salivary allergen of these insects. This 20-kDa protein (procalin) is a member of the lipocalin family, which includes salivary allergens from other invertebrates and mammals. An expression system capable of producing reagent quantities of recombinant allergen was developed in Saccharomyces cerevisiae. Antisera produced against recombinant protein cross-reacts with ELISA with salivary allergen. Recombinant Ag is also shown to react with sera from an allergic patient but not with control sera. By immunolocalization, the source of the salivary Ag is the salivary gland epithelium and its secretions.


Allergens/genetics , Allergens/immunology , Invertebrate Hormones/genetics , Invertebrate Hormones/immunology , Protein Precursors/genetics , Protein Precursors/immunology , Salivary Proteins and Peptides/genetics , Salivary Proteins and Peptides/immunology , Triatoma/genetics , Triatoma/immunology , Amino Acid Sequence , Anaphylaxis/immunology , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Gene Expression , Humans , Immunohistochemistry , Insect Bites and Stings/immunology , Insect Proteins , Lipocalins , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Saccharomyces cerevisiae/genetics , Saliva/immunology , Salivary Glands/immunology
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