A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a pro-inflammatory chemokine.

Chemokines are a family of chemotactic cytokines that regulate leukocyte migration. They are classified into four groups namely, CXC, CC, C and CX3C, based on the formation of a disulfide bridge. Among these, CXC chemokines have been identified as the largest group of chemokines in humans. In this study, we identified and functionally characterized a homolog of CXC chemokine from the big-belly seahorse, Hippocampus abdominalis, and designated it as ShCXCL. The cDNA of ShCXCL composed of a 342-bp open reading frame encoding 113 amino acids (aa). The CXC family-specific small cytokine domain (SCY) was identified from the mature peptide region, which comprised of a conserved CXC motif. As ShCXCL lacks an ELR (Glutamic acid-Leucine-Arginine) motif, it belongs to ELR- subfamily. The recombinant ShCXCL protein strongly induced the nitric oxide (NO) production in macrophage cells (RAW 264.7 cell line) and showed the chemotactic effect on flounder peripheral blood leukocytes. Tissue profiling showed a ubiquitous expression pattern in all examined tissues, with a high abundance in spleen. The up-regulated mRNA expression pattern of ShCXCL was observed in blood and kidney tissues after immune stimulation by live bacteria, such as Streptococcus iniae and Edwardsiella tarda, and mitogens, such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly I:C), suggesting its important role in host immune defense against microbial infection.

Thioredoxin domain-containing protein 12 from Oplegnathus fasciatus: Molecular characterization, expression against immune stimuli, and biological activities related to oxidative stress.

Thioredoxin domain-containing protein 12 (TXNDC12) is a small, disulfide-containing protein that belongs to the thioredoxin (TXN) superfamily. In the present study, we identified and characterized a TXNDC12-like gene, designated OfTXNDC12, from rock bream, Oplegnathus fasciatus. OfTXNDC12 consists of seven exons interrupted by six introns. Comparative genomic structural analysis revealed that the TXNDC12 of vertebrates is a structurally conserved gene. The coding sequence of OfTXNDC12 comprises 522 bp, which encodes 173 amino acid residues with the conserved thioredoxin active site motif, CGAC, and a probable C-terminal ER retrieval motif, GDEL. Transcriptional analysis of OfTXNDC12 showed the highest concentrations of the mRNA transcript in the liver, implying that it has a significant role in the liver under normal physiological conditions. In comparison, injection of lipopolysaccharide, Edwardsiella tarda, Streptococcus iniae, polyinosinic:polycytidylic acid (poly[I:C]) and rock bream iridovirus mostly triggered greater upregulation of OfTXNDC12 transcript levels in liver than in gill tissue, supporting its potential functional importance in the liver. Insulin disulfide reduction assay showed that the recombinant fusion protein (rOfTXNDC12) possesses significant thioredoxin activity. Treatment of LNCaP cells with the recombinant protein along with H2O2 revealed that rOfTXNDC12 increased the viability of cells and further supported its putative antioxidant capacity. Taken together, the results from our study suggest that OfTXNDC12 encodes for a potent antioxidant involved in redox regulation that shows significant responses to immune stimuli.

Identification and molecular characterization of peroxiredoxin 6 from Japanese eel (Anguilla japonica) revealing its potent antioxidant properties and putative immune relevancy.

Peroxiredoxins (Prdx) are thiol specific antioxidant enzymes that play a pivotal role in cellular oxidative stress by reducing toxic peroxide compounds into nontoxic products. In this study, we identified and characterized a peroxiredoxin 6 counterpart from Japanese eel (Anguilla japonica) (AjPrdx6) at molecular, transcriptional and protein level. The identified full-length coding sequence of AjPrdx6 (669 bp) coded for a polypeptide of 223 aa residues (24.9 kDa). Deduced protein of AjPrdx6 showed analogy to characteristic structural features of 1-cysteine peroxiredoxin sub-family. According to the topology of the generated phylogenetic reconstruction AjPrdx6 showed closest evolutionary relationship with Salmo salar. As detected by Quantitative real time PCR (qPCR), AjPrdx6 mRNA was constitutively expressed in all the tissues examined. Upon the immune challenges with Edwardsiella tarda, lipopolysaccharides and polyinosinic:polycytidylic acid, expression of AjPrdx6 mRNA transcripts were significantly induced. The general functional properties of Prdx6 were confirmed using purified recombinant AjPrdx6 protein by deciphering its potent protective effects on cultured vero cells (kidney epithelial cell from an African green monkey) against H2O2-induced oxidative stress and protection against oxidative DNA damage elicited by mixed function oxidative (MFO) system. Altogether, our findings suggest that AjPrdx6 is a potent antioxidant protein in Japanese eels and its putative immune relevancy in pathogen stress mounted by live-bacteria or pathogen associated molecular patterns (PAMPs).

Mitochondrial peroxiredoxin 3 (Prx3) from rock bream (Oplegnathus fasciatus): immune responses and role of recombinant Prx3 in protecting cells from hydrogen peroxide induced oxidative stress.

Pathogenic infections and environmental factors cause a variety of stresses in fish including oxidative stress by rapid elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Transcriptional activation and expression of antioxidant enzymes are essential for reducing the oxidative stress. In this study, we present the molecular characterization, immune responses and ROS scavenging activity of mitochondrial peroxiredoxin 3 from Oplegnathus fasciatus (RbPrx3). Coding sequence (CDS) of RbPrx3 contains 248 amino acids polypeptide which consists of highly conserved peroxiredoxin super family domain and two cysteine residues. Pairwise sequence comparison revealed that RbPrx3 has the greatest identity (94.8%) to Sparus aurata Prx3. Transcriptional analysis of RbPrx3 indicated the ubiquitously expressed mRNA in wide array of organs showing the highest expression in the liver of rock bream. Upon immune challenge of Edwardsiella tarda, Streptococcus iniae, rock bream iridovirus (RBIV) and lipopolysaccharide (LPS), RbPrx3 mRNA level was up-regulated in immunocompetent liver tissues compared to unchallenged fish. Purified recombinant RbPrx3 treated THP-1 cells showed higher survival rate against H(2)O(2) induced oxidative stress and significantly reduced the level of intracellular ROS. Overall results from our study suggest that RbPrx3 may be involved in broader functions such as regulating oxidative stresses by scavenging ROS and activating immune responses in rock bream.

Molluscan death effector domain (DED)-containing caspase-8 gene from disk abalone (Haliotis discus discus): molecular characterization and expression analysis.

The caspase family represents aspartate-specific cysteine proteases that play key roles in apoptosis and immune signaling. In this study, we cloned the first death effector domain (DED)-containing molluscan caspase-8 gene from disk abalone (Haliotis discus discus), which is named as hdCaspase-8. The full-length hdCaspase was 2855 bp, with a 1908 bp open reading frame encoding 636 amino acids. The hdCaspase-8 had 72 kDa predicted molecular mass with an estimated isoelectric point (PI) of 6.0. The hdCaspase-8 amino acid sequence contained the characteristic feature of an N-terminal two DED, a C-terminal catalytic domain and the caspase family cysteine active site 5¹³KPKLFFLQACQG5²4. Phylogenetic analysis results showed that hdCaspase-8 is more similar to the invertebrate Tubifex tubifex (sludge worm) caspase-8. Real-time RT-PCR results showed that hdCaspase-8 constitutively and ubiquitously expressed in all tested tissue of unchallenged disk abalone. The basal expression level of hdCaspase-8 in gill tissue was higher than all other tested tissues. The hdCaspase-8 mRNA expression in gill and hemocytes was significantly up-regulated by exposure to bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Listeria monocytogenes) and VHSV (viral hemorrhagic septicemia virus), as compared to control animals. These results suggest that hdCaspase-8 may be involved in immune response reactions in disk abalone.

Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): molecular characterization, cloning and expression analysis.

Heparin cofactor (HCII) is a serine protease inhibitor (SPI), and plays important physiological roles in various biological events including hemostasis. The gene encoding the HCII was isolated from GS-FLX™ genomic data of rock bream (Oplegnathus fasciatus), designated as RbHCII. The RbHCII (1950 bp) consists of a 1512 bp open reading frame (ORF) encoding 504 amino acids (aa), with a signal peptide of 19 aa residues. The predicted molecular mass and the estimated isoelectric point of RbHCII were 58 kDa and 5.9, respectively. The deduced aa sequence of RbHCII displayed a characteristic serpin domain and a serpin signature motif (FTVDQPFLFLI). RbHCII demonstrated homology with vertebrate HCIIs and the greatest degree of similarity (90.1%) was observed with Gasterosteus aculeatus HCII. Various functional domains including the reactive center loop (RCL), glycosaminoglycan (GAG) and thrombin binding sites and acidic repeats of human and RbHCII were found to be orthologs through the molecular modeling studies. Phylogenetic analysis revealed that RbHCII belongs to the clade D serpins, and is closely related to the clade A members. Constitutive expression of RbHCII mRNA was detected at different levels in various tissues in a tissue-specific manner. Interestingly, RbHCII transcription was significantly downregulated (p < 0.05) in liver after challenge with lipopolysaccharide (LPS), Edwardsiella tarda and rock bream iridovirus (RBIV). However, after the immune challenges, RbHCII showed a significant downregulation in blood tissue only at the late-phase of investigation. The recombinant RbHCII (rRbHCII) was overexpressed in Rosetta-gami (DE3) cells and purified using the pMAL™ system. The rRbHCII inhibited thrombin and chymotrypsin in a dose-dependent manner. Remarkably, heparin was found to be an enhancer of RbHCII's thrombin-inhibitory activity. Correlating the heparin-dependent thrombin-inhibition activity of RbHCII with its temporal downregulation against immune stimulants, it could be suggested that it is not only involved in the blood coagulation cascade, but also plays an incognito role in immune modulation.

Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream (Oplegnathus fasciatus).

Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca(2+) binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.

BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone.

Allograft inflammatory factor-1 in disk abalone (Haliotis discus discus): molecular cloning, transcriptional regulation against immune challenge and tissue injury.

Here, we report the identification and characterization of allograft inflammatory factor-1 (AIF-1) from disk abalone Haliotis discus discus that was denoted as AbAIF-1. The full-length cDNA of AbAIF-1 consists of a coding region (453 bp) for 151 amino acids with a 17 kDa molecular mass. Analysis of AbAIF-1 sequence showed that it shares characteristic two EF hand Ca(+2)-binding motifs. Results from phylogenetic analysis further confirm that AbAIF-1 is a member of the AIF-1 family similar to invertebrate and vertebrate counterparts suggesting it has high evolutional conservation. Tissue-specific expression and transcriptional regulation of AbAIF-1 were analyzed after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes), viral hemorrhagic septicemia virus (VHSV) immune challenge and during tissue injury by quantitative real-time PCR. It is shown that the expression of AbAIF-1 mRNA was expressed ubiquitously in all selected tissues in constitutive manner showing the highest level in hemocytes. Upon bacteria and VHSV challenge, AbAIF-1 showed the significant up-regulation in hemocytes than gills. After the tissue injury in shell and mantle, AbAIF-1 and antioxidant selenium-dependant glutathione peroxidase (SeGPx) transcripts were significantly upregulated in abalone hemocytes. Taken together, these findings suggest that AIF-1 could response against the pathogenic challenge or tissue injury in abalone like mollusks. Also, AbAIF-1 may involve in wound healing and shell repair after the tissue injury of abalone.

A Brain Atlas of the Long Arm Octopus, Octopus minor.

Cephalopods have the most advanced nervous systems and intelligent behavior among all invertebrates. Their brains provide comparative insights for understanding the molecular and functional origins of the human brain. Although brain maps that contain information on the organization of each subregion are necessary for a study on the brain, no whole brain atlas for adult cephalopods has been constructed to date. Here, we obtained sagittal and coronal sections covering the entire brain of adult Octopus minor (Sasaki), which belongs to the genus with the most species in the class Cephalopoda and is commercially available in East Asia throughout the year. Sections were stained using Hematoxylin and Eosin (H&E) to visualize the cellular nuclei and subregions. H&E images of the serial sections were obtained at 30~70-µm intervals for the sagittal plain and at 40~80-µm intervals for the coronal plain. Setting the midline point of the posterior end as the fiducial point, we also established the distance coordinates of each image. We found that the brain had the typical brain structure of the Octopodiformes. A number of subregions were discriminated by a Hematoxylin-positive layer, the thickness and neuronal distribution pattern of which varied markedly depending upon the region. We identified more than 70 sub-regions based on delineations of representative H&E images. This is the first brain atlas, not only for an Octopodiformes species but also among adult cephalopods, and we anticipate that this atlas will provide a valuable resource for comparative neuroscience research.