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Abstract: Nitric oxide and secondary oxides of nitrogen react with unsaturated fatty acids such as linoleic acid to yield oxidized and nitrated products. Fatty acid nitroalkene derivatives, (e.g. nitrolinoleate [LNO2]) are produced by oxidative inflammatory reactions, detected clinically, display potent electrophilic reactivity and induce post-translational protein modifications that mediate adaptive inflammatory signaling responses. LNO2 signaling was examined in lung epithelial cells because the alveolar compartment is a rich site for the transduction of redox and inflammatory reactions. LNO2 did not directly induce Ca2+ influx in cultured lung epithelial cells, but inhibited bradykinin-induced Ca2+ influx in a cGMP-independent manner. In contrast, LNO2 activated MAP kinase (Erk1/2) by a mechanism independent of bradykinin. It was hypothesized that these unique responses were transduced by activation of different protein kinase C isotypes, supported by the observation that LNO2-mediated inhibition of Ca2+ influx was blocked by the non-selective PKC inhibitors chelerythine chloride and calphostin C, but not by the calcium dependent “classic” PKC inhibitor Gö6976. Western blot analysis showed that atypical PKCζ was activated by LNO2 stimulation, with PKCζ and Erk activation also demonstrated in primary culture of human lung type II cells. Addition of pseudotypical PKCζ substrate peptide reversed LNO2-mediated induction of Ca2+ influx and MAP kinase activation. Finally, the electrophilic nature of LNO2 resulted in a novel mode of PKCζ activation, covalent adduction of the enzyme. In summary, LNO2 mediated signaling in lung type II epithelial cells occurs via a unique pathway involving PKCζ. [Copyright &y& Elsevier]

Abstract: The JAK/STAT signal transduction pathway plays a critical role in host defence against viral and bacterial infections. In the present study, we report cDNA cloning and characterization of the JAK family (mJAK1-3 and mTYK2) and STAT family members (mSTAT1, mSTAT3-6) from the mandarin fish Siniperca chuatsi. To our knowledge, JAK2, TYK2 and STAT6 genes were cloned from fish for the first time. The mJAK family proteins consist of 1112–1177 residues with a FERM domain, an SH2 domain, a pseudokinase domain, and a tyrosine kinase domain. The mSTAT family members contain 716–786 residues with similar architecture, including an N-terminal domain, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a transcription activation domain. Multiple sequence alignments of mJAKs/mSTATs and phylogenetic analysis showed that mJAK1 was closed to mTYK2, and mJAK2 was closed to mJAK3. Quantitative real-time PCR results revealed that mJAK/mSTAT family members were expressed in most tissues examined except muscle. In mandarin fish fry cells, the expressions of IRF-1, Mx, SOCS1 and SOCS3 genes were significantly induced by poly(I:C) stimulation, indicating that the mJAK/mSTAT signal pathway is activated by poly(I:C). Furthermore, expressions of all four mJAKs and four mSTATs were all up-regulated after poly(I:C) stimulation, but expression of mSTAT5 was inhibited by poly(I:C). These results suggest that mandarin fish has the JAK/STAT signal transduction pathways similar to those in mammals, and these signalling pathways may play an important role in regulation of antiviral responses in fish. [Copyright &y& Elsevier]

Abstract: The NF-κB/IκBα pathway plays an important role in the regulation of immune and inflammatory responses. IκBα is an inhibitory molecule that sequesters transcription activator NF-κB dimer in the cytoplasm of unstimulated cells. Here, we isolated the full-length cDNAs of the mandarin fish (Siniperca chuatsi) alpha inhibitor of NF-κB (ScIκBα) and p65 NF-κB (Scp65). Multiple sequence alignments showed that the amino acid sequences of both ScIκBα and Scp65 contain conserved domains similar to those of mammalian counterparts. Protein pull-down and coimmunoprecipitation assays showed that ScIκBα directly bound with Scp65. Real-time quantitative PCR analysis showed that ScIκBα mRNA was constitutive in all mandarin fish tissues detected. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the mRNA level of ScIκBα was decreased nearly 6 fold in the spleen. This result suggests that the NF-κB/IκBα pathway in mandarin fish may play a role in the immune response against ISKNV. [Copyright &y& Elsevier]