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Chemokine (C-X-C motif), receptor 4 (CXCR4), a member of the family of seven transmembrane G-protein-coupled receptors, plays important roles in immunomodulation, organogenesis, hematopoiesis, and derailed cerebellar neuron migration. We characterized the sequences and expression profiles of CXCR4b in Megalobrama amblycephala. The full-length cDNA was 1638bp, encoding 353 amino acid residues. Multiple alignment and phylogenetic analysis indicated that M. amblycephala CXCR4b contained the similar conservative sequences and motifs with other organisms. The CXCR4b expression in different development stages of M. amblycephala showed the mRNA levels before hatching and at 62h post fertilization (hpf) were significantly higher than at other post hatching stages (P<0.05). Besides, CXCR4b was constitutively expressed in a wide range of tissues, at higher levels in headkidney, liver, intestine spleen, blood and gill, where a larger number of immune cells including lymphocytes and macrophages reside, suggesting its specific roles in inflammatory responses. The CXCR4b expression after high nitrite concentration (ρNO(2-)-N: 20.29mg/L) exposure supported a potential pro-inflammatory function for CXCR4b. In order to identify the better population with immune property for breeding, we compared the tissue expression of CXCR4b among Liangzi Lake population (L), Yuni Lake population (Y) and Poyang Lake population (P), it was indicated that the expression levels in the population Y were obviously higher than that of the other two populations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5 , PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates. • A total of 38 ABC genes were systematically identified and characterized in P. vannamei genome. • Most PvABC genes were ubiquitously expressed in all examined tissues with high expression in hemocytes, gill, hepatopancreas and intestine. • The mRNA expression of selected PvABC genes was obviously altered after V. parahemolyticus challenge. • PvABCC5 and PvABCF1 knockdown increased shrimp mortality and the bacteria loads in the hepatopancreas upon V. parahemolyticus challenge. • PvABCC5 and PvABCF1 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway, and then promoting the expression of AMPs in P. vannamei. [ABSTRACT FROM AUTHOR]

Abstract HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation. Highlights • Megalobrama amblycephala phd1 was not induced by hypoxia in most tissues. • Two Phd1 isoforms generated by alternative initiation were identified in the nucleus. • Only the senior isoform promoted cell proliferation. [ABSTRACT FROM AUTHOR]

PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3 . Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala . [ABSTRACT FROM AUTHOR]

Abstract: Cardiac troponin T (TNNT2), as a member of troponin superfamily, plays important roles during early cardiogenesis, and contraction and relaxation of myocardial cells. In this study, two alternatively spliced variants of Megalobrama amblycephala TNNT2 were identified showing a difference of 19 amino acids in the N-terminal hypervariable region. The longer cDNA (TNNT2-1) was 1118bp, encoding 284 amino acid residues, contained conserved central tropomyosin-binding region, cardiac specific signal and C-terminal segments except the N-terminal hypervariable region. The TNNT2 transcripts first appeared at 16hours post-fertilization (hpf) peaking at 28hpf (onset of heartbeat). In addition, strong expression of TNNT2 was found in the cardiac muscle. After nitrite exposure, the increased TNNT2 expression levels in the heart indicated that nitrite might induce cardiac injury. Results of semi-quantitative RT-PCR indicated that the two alternatively spliced variants existed in early development stages since their first appearance at 16hpf and heart, spleen, headkiney of M. amblycephala. The shorter transcript (TNNT2-2) was proved to be dominant in the embryos and heart of M. amblycephala, furthermore, the increase of TNNT2 expression level in the heart after nitrite exposure was mainly caused by TNNT2-2. [Copyright &y& Elsevier]

Abstract: Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in oxygen tension in the cellular environment. In this study, we identified full-length cDNAs of HIF-1α, HIF-2α and HIF-3α in an endangered hypoxia-sensitive fish species, Chinese sucker. The HIF-1α/2α/3α cDNAs are 3890, 3230 and 3374bp in length, encoding 780, 782 and 632 amino acid residues, respectively. The real-time PCR results suggested that HIF-1α and HIF-3α mRNAs were highly expressed in liver and gonads, followed by spleen and muscle. Moreover, HIF-1α and HIF-3α transcription factors revealed similar developmental expression patterns, with the lowest expression at 48h post-fertilization, and reaching the highest expression level at 360h post-fertilization. Short-term hypoxia exposure (2.2, 2.8 and 3.2mg/L dissolved oxygen for 24h) increased mRNA levels of HIF-1α and HIF-3α. HIF-2α mRNA showed similar expression patterns with that of HIF-1α and HIF-3α, however, its expression was extremely low in the spatio-temporal expression patterns and hypoxia treatment. This is the first report describing the potential to identify hypoxia-sensitive/tolerant fishes according to the number of the serine residues of fish oxygen-dependent degradation (ODD) domain. It was suggested that Cyprinomorpha fishes, with less than 40 serine residues in fish ODD domain were hypoxia-sensitive fishes and more than 40 serine residues in this domain were hypoxia-tolerant fishes. [Copyright &y& Elsevier]

Abstract: This study examined the gene structures and expression of trypsinogens, as well as the trypsin activities of the grass carp Ctenopharyngodon idellus (herbivorous) and the topmouth culter Culter alburnus (carnivorous), which are commercially important freshwater species of the family Cyprinidae in China. Isolated full-length trypsinogen cDNA clones were 869bp and 857bp. The deduced amino acid sequences were 242aa and 247aa long, both containing the highly conserved residues essential for serine protease catalytic and conformational maintenance. The results from isoelectric and phylogenetic analyses suggest that grass carp trypsinogen is grouped with teleost trypsinogen group I, while topmouth culter trypsinogen is grouped with group II. The expression pattern of trypsinogen mRNA was similar between these two species, appearing 2days post-hatching (dph) and reaching peaks at 11 and 23dph. The trypsin-specific activities in both species were detected 2dph and reached the major peaks at 8dph, however the minor peaks were observed at 20dph in the grass carp and 17dph in the topmouth culter. The trypsin-specific activity was significantly higher in the grass carp than in the topmouth culter, which may be attributed to the nature of their different nutritional habits. [Copyright &y& Elsevier]