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Extracellular ATP is an important damage-associated molecular pattern molecule that plays key roles in innate immunity. In fish, however, the mechanism for extracellular ATP release remains largely undefined. Pannexin1 (Panx1) is a newly discovered extracellular ATP release channel with a wide tissue distribution and diverse biological functions in mammals. In the present study, we identified and characterized a Panx1 homolog cDNA, termed poPanx1 , from Japanese flounder Paralichthys olivaceus , which is one of the most important economic mariculture fish species in China. PoPanx1 is a membrane protein that is composed of 437 amino acids with an estimated molecular mass of 48.7 kDa and an isoelectric point of 6.46. The poPanx1 mRNA ubiquitously expresses in all examined tissues but with predominant expression in hepatopancreas in unstimulated healthy adult Japanese flounder. In Japanese flounder head kidney primary cells, poPanx1 gene expression could be significantly induced by pathogen-associated molecular patterns (PAMPs; polyinosinic–polycytidylic acid and bacterial endotoxin LPS) stimulations. In vivo experiments revealed that poPanx1 mRNA expression was significantly up-regulated upon immune challenges with Edwardsiella tarda and Vibrio anguillarum . Furthermore, we showed that poPanx1 is an important channel protein for PAMP-induced extracellular ATP release that is required for activation of purinergic signaling in fish innate immunity. Taken together, our findings suggest that the ATP release channel, poPanx1 , is a novel immune response gene in purinergic signaling of Japanese flounder P . olivaceus . [ABSTRACT FROM AUTHOR]

Abstract: Lipopolysaccharide-induced TNF-α factor (LITAF) is an important transcription factor participating in innate immunity through regulating TNF-α and other inflammatory cytokines expression. However, the expression and biological relevance of LITAF in fish is still very limited. In this study, a full-length LITAF cDNA, termed PoLITAF, was identified from Japanese flounder Paralichthys olivaceus. PoLITAF contains a 67bp 5′-untranslated sequence, a 435bp open reading frame, and a 647bp 3′-untranslated sequence. PoLITAF protein is comprised of 144 amino acids with a conserved C-terminal LITAF-like domain and shows 51–76% sequence similarity and 40–65% sequence identity with other LITAF homologues. Characterization of this new gene revealed that PoLITAF mRNA was detected in all examined tissues with the highest expression in gill. In head kidney primary culture, the expression of Japanese flounder PoLITAF and TNF-α was significantly up-regulated in response to Poly(I:C) and bacterial endotoxin LPS stimulation. Further in vivo experiments demonstrated that PoLITAF expression was up-regulated in head kidney, gill and spleen post bacterial challenge with Edwardsiella tarda. Moreover, the up-regulated expression of Japanese flounder TNF-α following the enhanced expression of PoLITAF was detected as early as 4h in both gill and head kidney tissues and 12h in spleen after the bacterial infection in vivo. Our findings suggest that PoLITAF is a novel inducible gene possibly involved in Japanese flounder innate immunity. [Copyright &y& Elsevier]

P2X4 receptor (P2X4R) is a member of trimeric ATP-gated receptor channel family. Despite the importance of P2X4R in innate immunity has been addressed in mammals, the immunological significance of P2X4R has not been characterized in fish. In the present study we identified a full-length P2X4R cDNA sequence from Japanese flounder Paralichthys olivaceus (termed poP2X4R ) by RT-PCR and RACE approaches and analyzed its gene expression patterns under normal and immune challenge conditions. Qualitative RT-PCR analyses revealed that poP2X4R has a widespread distribution in all examined tissues but dominantly expressed in hepatopancreas. In Japanese flounder head kidney macrophages and peripheral blood lymphocytes, poP2X4R was rapidly and significantly up-regulated by the immune challenges of LPS, poly(I:C) and zymosan. In addition, poP2X4R was up-regulated in spleen, head kidney and gill tissues by Edwardsiella tarda infections. Furthermore, we showed that poP2X4R is a membrane glycoprotein which could interact with ATP release channel Pannexin1, an important component in extracellular ATP-activated purinergic signaling pathways involved in Japanese flounder innate immune response. From a comparative immunological point of view, our results have provided new evidence for the involvement of extracellular ATP-gated P2XRs in fish innate immunity. [ABSTRACT FROM AUTHOR]

Abstract: Lipopolysaccharide-induced TNF-alpha factor (LITAF) is an important transcription factor in transcriptional regulation of TNF-alpha and other cytokines. Here we identified a full-length LITAF homolog cDNA, termed EsLITAF, that contains a 71 bp 5′-untranslated sequence, an open reading frame consisting of 381 bp, and a 208 bp 3′-untranslated sequence in Chinese mitten crab (Eriocheir sinensis), an economically important freshwater crustacean species in China. EsLITAF protein possesses a conserved C-terminal LITAF-like domain with two CXXC motifs and is comprised of 126 amino acids with a theoretical molecular mass of 13.1 kDa and an isoelectric point of 6.36. Blast search against GenBank database revealed that EsLITAF protein shared the highest sequence identity (84%) with the counterpart of Pacific white shrimp (Litopenaeus vannamei). EsLITAF mRNA transcript expresses ubiquitously in all examined tissues with the highest expression in hepatopancreas and lowest expression in haemocytes. Furthermore, EsLITAF mRNA expression could be significantly and rapidly induced in haemocytes by LPS and Poly(I:C) stimulations in vitro. Moreover, EsLITAF gene expression was up-regulated in haemocytes, gill and hepatopancreas after Edwardsiella tarda and Vibrio anguillarum challenges. Taken together, we have identified and characterized a new crustacean LITAF homolog from the Chinese mitten crab. [Copyright &y& Elsevier]