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The sea cucumber Apostichopus japonicus is dioecious, with seasonal reproduction. G protein-coupled receptor (GPCR)-mediated signaling systems might play critical roles in the reproductive control of A. japonicus. Here, we classified GPCR from the genome in silico and used transcriptomic analyses to further mine those that function in gonadal-development control. Totally, 487 GPCRs were predicted from A. japonicus, and 183 of these were further annotated to molecular pathways. Transcriptome analysis revealed 327 GPCRs expressed in gonads, and these were classified into four families and 19 subfamilies. Three pathways were apparently associated with reproduction, including neuroactive ligand-receptor interaction, the mTOR and Wnt signaling pathways. Seven and eight ovary- and testis-specific GPCRs were filtered, and the gene expression profiles were determined in multiple tissues and gonads at different developmental stages by qPCR. These results provide new insights into the discovery of GPCR-mediated signaling control in sea cucumber reproduction, especially in gonadal development control.

Interleukin-8 (IL-8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by multiple cell types. It promotes chemotaxis and phagocytosis via interaction with chemokine receptors CXCR1 and CXCR2. Using published data, IL-8 gene (LcIL-8) of the large yellow croaker (Larimichthys crocea) was cloned into the pcDNA3.1 plasmid, and an interleukin-8 receptor (LcCXCR2) was cloned into the pEGFP-N1 plasmid. Secratory expression of LcIL-8 in HEK293T cells was carried out, and product in culture medium was collected for LcCXCR2 stimulation in HEK293 cells. Following receptor internalization observation and intracellular signaling detection, the functional interaction of LcIL-8 and LcCXCR2 was further determined and the ERK phosphorylation signal activation mediated by LcCXCR2 was demonstrated. Quantitative real-time PCR analysis was used to analyze transcription level regulation of LcIL-8 and LcCXCR2 in various tissues of large yellow croaker. Expression of LcIL-8 and LcCXCR2 was elevated in the spleen, head kidney, and liver after Vibrio parahemolyticus challenge. Results illustrated the functional interaction between LcIL-8 and LcCXCR2 in mediating intracellular ERK1/2 phosphorylation signaling and suggested that the LcIL-8 and LcCXCR2 system is part of the immune response induced by V. Parahemolyticus in L. crocea.

Interleukin-8 (IL-8, CXCL8), a pro-inflammatory chemokine secreted by a variety of cell types, plays a critical role in the development of various immune diseases. Interactions between IL-8 and its receptor CXC receptor 1/2 (CXCR1/2) are known to promote chemotaxis and phagocytosis in many immune responses. In this study, we report the molecular characteristics and pharmacological activity of CXCR1 (MsCXCR1) in largemouth bass (Micropterus salmoides) and evaluated the functional involvement of MsCXCR1 in individuals infected with the pathogen Nocardia seriolae. MsCXCR1 was cloned into the pEGFP-N1 plasmid and the subcellular localization of MsCXCR1 on the cell membrane was verified in MsCXCR1-EGFP-expressing HEK293 cells. Following observation of receptor internalization and intracellular signaling detection, we further determined the functional interaction of secreted interleukin-8 (LcIL-8, the ligand for CXCR1 in large yellow croaker) and MsCXCR1 was further determined, and the ERK phosphorylation signal activation mediated by MsCXCR1 was demonstrated. Quantitative real-time PCR assays were conducted to analyze the transcriptional distribution of MsCXCR1 in various tissues of healthy and diseased largemouth bass. These results illustrate the significant elevation of MsCXCR1 expression in the head kidney, spleen and liver of M. salmoides, suggesting that MsCXCR1 was involved in the immune response in N. seriolae-infected largemouth bass and potentially affects the digestive function of this species.

Serotonin (5-hydroxytryptamine [5-HT]) receptors (5-HTRs) mediate neuroendocrine signaling via interactions with the ligand serotonin (5-HT). The 5-HT signaling system has been well studied in vertebrates, but rarely known in invertebrate animals, especially in the marine invertebrates. In this study, we identified and characterized a novel 5-HTR from the sea cucumber Apostichopus japonicus (Aj5-HT4/6 ). The cloned Aj5-HT4/6 open reading frame comprised 1290 bp and encoded 429 amino acids. Bioinformatic analysis of the receptor indicated that it was a member of the class A of the G protein-coupled receptor family. Further experiments using Aj5-HT4/6 -transfected HEK293 cells demonstrated that treatment with 5-HT could induce rapid internalization of Aj5-HT4/6 fused with enhanced green fluorescent protein from the cell surface into the cytoplasm and triggered a significant increase in levels of the second messenger cAMP as well as mitogen-activated protein kinase phosphorylation in a 5-HT dose-dependent manner. Quantitative real time-polymerase chain reaction demonstrated that Aj5-HT4/6 was predominantly expressed in the muscle and respiratory tree, and its expression was significantly decreased during estivation. Taken together, these results imply that Aj5-HT4/6 is potentially involved in the movement and metabolism of the sea cucumber.

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

CCHamides are newly identified insect neuropeptides, which are widely occurring in most insects. However, our knowledge about their signaling characteristics and physiological roles is still limited. Here, we cloned two full-length cDNAs encoding putative CCHamide receptors, Bombyx neuropeptide GPCR A14 (BNGR-A14) and -A15 (BNGR-A15), from the brain of B. mori larvae. Characterization of signaling indicated that Bombyx CCHamide-1 and CCHamide-2 are specific endogenous ligands for BNGR-A15 and BNGR-A14, respectively. Further functional assays combined with specific inhibitors demonstrated that upon activation by CCHamide-2, BNGR-A14 elicited significant increases in CRE-driven luciferase activity, intracellular Ca2+ mobilization and ERK1/2 phosphorylation in a Gq inhibitor-sensitive manner, while BNGR-A15 was activated by CCHamide-1, thus leading to intracellular accumulation of cAMP, Ca2+ mobilization, and ERK1/2 phosphorylation in a Gs and Gq inhibitor-sensitive manner. Based on these findings, we designated the receptors BNGR-A15 and -A14 as Bommo-CCHaR-1 and -2, respectively. In addition, our results showed that CCHamides are considered to require intrachain disulfide bonds to activate their respective receptor in the physiological concentration range. Moreover, quantitative RT-PCR analysis revealed that CCHamide-1 is more likely to serve as a brain-gut peptide to regulate feeding behavior and growth through BNGR-A15, whereas the CCHamide-2 signaling system might play an important role in the control of multiple physiological processes. Our findings provide in-depth information on CCHamide-1 and -2-mediated signaling, facilitating further elucidation of their endocrinological roles in the regulation of fundamental physiological processes.

Gonadotropin-releasing hormone (GnRH) plays a vital role in the regulation of reproduction through interaction with a specific receptor (the GnRH receptor). In this study, the GnRH receptor gene from the cuttlefish Sepiella japonica (SjGnRHR) was identified and characterized. The cloned full-length SjGnRHR cDNA was 1,468 bp long and contained a 1,029 bp open reading frame encoding 342 amino acid residues, 8 bp of 5' untranslated regions (UTR), and 431 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 38.75 kDa and an isoelectric point of 9.47. In addition, this protein was identified as belonging to the rhodopsin-type (class A) G protein-coupled receptor family. The predicted amino acid sequence contained two N-linked glycosylation sites and 18 phosphorylation sites. Multiple sequence alignment, phylogenetic tree analysis, and three-dimensional structure modeling were conducted to clarify SjGnRHR bioinformatics characteristics. In vitro SjGnRHR expression was carried out using HEK293 cells and the pEGFP-N1 plasmid, to verify the transmembrane properties of this protein. The interaction between the S. japonica GnRH receptor and its ligand was clarified using internalization analysis. SjGnRHR transcriptional quantification confirmed the wide distribution of SjGnRHR in various S. japonica mature tissues. In addition, the transcriptional profile of SjGnRHR in the female brain and ovary during gonadal development was analyzed. Results indicate that GnRHR may be involved in diverse S. japonica physiological functions, especially in the control of reproduction.

Sex steroid hormones are widely detected in molluscs and play important roles in sex determination, gonadal tissue maturation, and gametogenesis. Nevertheless, the signaling pathways of sex steroids in cephalopod have not yet been clearly elucidated. In the present study, a full-length sequence encoding the estrogen receptor (ER) was isolated from common Chinese cuttlefish, Sepiella japonica. The sjER cDNA clone was found to contain 1,788 nucleotides including a 1,470 bp open reading frame encoding 489 amino acid (aa) residues. The deduced ER protein consisted of six nuclear receptor characteristic domains. Based on a phylogenetic analysis, the ER DNA-binding domain and ligand-binding domain are highly conserved compared to other mollusc ERs. Highest aa identities were found for sjER with common octopus (Octopus vulgaris) ER (89%) and pacific oyster (Crassostrea gigas) ER (61%). Tissue expression analysis confirmed that sjER was widely distributed among tissues and predominantly expressed in the brain, liver, gonad (testis and ovary), and other accessory sexual gland (nidamental gland). The ER expression was temporally upregulated in the brain, liver, and ovary during the early sexual maturation period in S. japonica, which is coincident with the fluctuation of ovary estradiol content. These suggest that sjER may be involved in regulating the reproductive cycle of S. japonica. A fusion protein transient transfections assay showed that sjER was mainly located in the nucleus, suggesting a possible orthodox working mechanism of S. japonica ER in the nucleus through a ligand-dependent activation of specific gene transcription.

Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5' UTR and a 599 bp 3' UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133-144, 278-288 and 295-314. Analysis of the predicted tertiary structure of prepro-CatL (17-333 aa) and mature-CatL (116-333 aa) reveals that the propeptide region (17-115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response.

Neuropeptide Y (NPY) receptors are suggested to mediate the multi-physiological functions of NPY family peptides, such as food intake, in teleost fish. However, the structure and signaling of fish NPY receptors are yet to be fully elucidated. In this study, we report the cloning and characterization of two neuropeptide Y receptor subtypes, Y2 (NPY2R) and Y7 (NPY7R), in yellow croaker Larimichthys crocea (L. crocea) (LcNPY2R, LcNPY7R). The gene structure, pharmacological characterization, cell location, and tissue expression of these two receptors were explored. The phylogenetic results showed that LcNPY2R and LcNPY7R had typical G protein-coupled receptor profiles, associated with the Y2 subfamily, with coding sequences that are highly conserved in vertebrates. The expression of both LcNPY2R and LcNPY7R could be activated by LcNPY in HEK293 cells. However, truncated LcNPY18-36 was only able to activate LcNPY2R at the same level as full length LcNPY. Expression analysis revealed that LcNPY2R mRNA was predominantly expressed in the intestine and liver, whereas LcNPY7R was expressed in the stomach, which indicated that both receptors were related to the digestive system. Overall, our data establishes a molecular basis to determine the actions of LcNPY2R and LcNPY7R, which could be used to elucidate the conserved roles of these receptor-ligand pairs in vertebrates.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

Aestivation is a state of dormancy in diverse animal species characterized by a depressed metabolism. High temperatures in the summer trigger aestivation in the adult sea cucumber, Apostichopus japonicus. In the present study, the glycolytic suppression in aestivating A. japonicus was verified by metabolite quantification and activity and a gene expression analysis of rate-limiting enzymes (hexokinase and pyruvate kinase). Significant decreases in glucose concentrations were discovered in the respiration tree (p

Scavenger receptors (SRs) are crucial pattern recognition receptors (PRRs) to defense pathogen infection in fish innate immunity. In this paper, some members in SRs family of Larimichthys crocea were identified, including eight genes in the class A, B, D and F families. (G + C) % of all SRs members held 51% ∼ 59%, and these genes were no obvious codon bias by analyzing the distribution of A-, T-, G- and C-ended codons. The order of Enc for all SRs members by sequencing was LycCD68 > LycSCARA5 > LycSCARB1 > LycCD163 > LycMARCO > LycSREC1 > LycSCARA3 > LycSREC2. Moreover, different lengths and numbers of exons and introns led to the diverse mRNAs and respective functional domains or motifs, for example, an optional cysteine-rich (SRCR) domain in LycMARCO and LycSCARA5, an epidermal growth factor (EGF) and EGF-like domain in LycSREC1 and LycSREC2. The sub-cellular localization demonstrated SRs members mainly located in plasma membrane or extracellular matrix. Further, all of the SRs members in L. crocea were almost low expressed in heart, gill and intestine, whereas high in spleen and liver. After stimulation by Vibrio alginolyticus, the class A and F families were induced significantly, but the class B and D families expressed less even none after pathogenic infection. All the findings would pave the way to understand not only the evolution of the SR-mediated immune response, but also the complexity of fish immunity.

Gene therapy may be described as the use of genes as medicines to treat disease, or, more precisely, as the delivery of nucleic acids by means of vectors to patients for some therapeutic purpose (Thanou, M. et al., 2007). The major goal of gene therapy is to introduce a functional gene into a target cell and restore protein production that is absent or deficient due to a genetic disorder (Neeltje, A. et al., 2003). This approach is a potentially powerful method for the treatment of diseases for which classical pharmacotherapy is unavailable or not easily applicable. Gene therapy is a therapeutic modality with enormous promise, which is also considered to have failed to deliver much of therapeutic significance in spite of all the apparent clinical interest. Clinical trial activity in gene therapy began in 1989, peaked in 1999, and is now currently declining (Thanou, M. et al., 2007). This decline was marked by some clinical trial problems, including a death from toxic liver shock during an adenovirus-based clinical trial in 1999 (Marshall, E., 2000), the anomalous appearance of a transgene in the gonads during adeno-associated virus-based preclinical trials in 2001 (Arruda, V. R. et al., 2001), signs of hypertension in lipofection clinical trials in 2005 (Pro-1) (MacLachlan, I. et al., 1999), and the development of leukemia in retrovirus-based clinical trials for ex vivo treatment of X-linked severe combined immunedeficiency (X-linked SCID) (Cavazzana-Calvo, M. et al., 2004; Gaspar, H. B., & Thrasher, A. J., 2005). Lessons from those frustrated results suggest that more basic research is required in gene therapy study, including mechanism of diseases and features of viral vectors. In order to modify a specific cell type or tissue, the therapeutic gene must be efficiently delivered to the cell, so that it will express at the appropriate level for a sufficient duration. Thus, identifying the ideal means of carriage for viral gene therapy is the key rate-limiting step in the development of most promising gene therapy strategies. In spite of long-term and extensive efforts to develop in vivo gene delivery systems, little achievements have been reported, especially as far as clinical applications are concerned. Apparently, the development of gene delivery systems will be one of the most critical issues for the success of in vivo gene therapy. Over the years, two broad approaches have been used to deliver therapeutic genes to cells, viral vectors and non-viral vectors. These two kinds of vectors are different as regard to