Molecular cloning and expression of MyD88 in large yellow croaker, Pseudosciaena crocea.

Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). In this report, the full-length cDNA of MyD88 was cloned from the large yellow croaker, Pseudosciaena crocea. It was of 1574 bp, including a 5'-terminal untranslated region (UTR) of 89 bp, a 3'-terminal UTR of 621bp and an open reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids. It contained a typical death domain at the N-terminal and a conservative Toll/IL-1R (TIR) domain structure at the C-terminal. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of MyD88 with the highest expression in the spleen and the weakest expression in the muscle. The expression of MyD88 after challenge with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. It suggested that the highest expression was in the spleen (p<0.05) with 1.9 times (at 48 h) as much as that in the control and the lowest expression of MyD88 was in the liver (p<0.05) with 0.29 times (at 3h) of that in the control. These results indicated that as a universal key adaptor in the Toll-like receptor pathway in mammals, MyD88 might play an important role in large yellow croaker defense against pathogenic infection.

{[1-(Arylmethyl)piperidin-4-yl]oxy}-(trifluoromethyl)-pyridines: ketanserin analogues with insect growth regulating activity.

A series of {[1-(arylmethyl)piperidin-4-yl]oxy}-(trifluoromethyl)-pyridine derivatives were designed and synthesized on the basis of the ketanserin (1) framework, a prototypic mammalian 5-HT(2A) receptor antagonist, and the structure-activity relationship (SAR) was also discussed. The result of the bioassay showed that most of the title compounds inhibited the insect growth and exhibited moderate-to-good growth regulating activity against the armyworm Pseudaletia separata Walker. Furthermore, the SAR study revealed that, when the determinant feature, interacting with mammalian 5-HT(2A) receptor, was preserved, a simplified ArCH(2) group greatly contributed to insect growth inhibitory activities. It was also found that the substituted position of the CF(3) group at the pyridine ring played a key role, and that the introduction of 1-[bis(4-fluorophenyl)methyl]piperazine, an equivalent of the benzoylpiperidine moiety of ketanserin, resulted in bioactivities similar to those of the title compounds, which were in agreement with the model of ketanserin analogues binding to mammalian 5-HT(2) receptors.

Cloning and expression analysis of two alternative splicing toll-like receptor 9 isoforms A and B in large yellow croaker, Pseudosciaena crocea.

Toll-like receptors (TLRs) are the archetypal pattern-recognition receptors in sensing foreign pathogens. In this report, two alternative splicing isoforms of TLR9 (named PcTLR9A and PcTLR9B) cDNA were cloned from the large yellow croaker, Pseudosciaena crocea. The full-length cDNA of PcTLR9A was of 3637bp, including a 5'-terminal untranslated region (UTR) of 111bp, 3'-terminal UTR of 355bp and an open reading frame (ORF) of 3171bp encoding a polypeptide of 1056 amino acids. However, the full-length cDNA of PcTLR9B was 119bp longer than that of PcTLR9A from the position of 3079-3197bp, which encoded a polypeptide of 1006 amino acids. Both of the PcTLR9A and PcTLR9B contained 12 typical structures of leucine-rich repeats (LRRs), an LRRTYP and an LRRCT in the extracellular region and a conservative Toll/IL-1R (TIR) domain in the intracellular region. However, compared to PcTLR9A, conservative Box 3 was absent in PcTLR9B TIR domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR9A and PcTLR9B with the highest expression in spleen and the weakest expression in muscle. Expression of PcTLR9A and PcTLR9B after stimulation with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. This indicated that the highest expression was 3.3 times (at 24h) as much as that in the control in the spleen (p<0.05) and the lowest expression of PcTLR9A was 1/4 times (at 3h) of that in the control in the liver (p<0.05). PcTLR9B showed a similar expression pattern to PcTLR9A post-injection. These results suggested that both PcTLR9A and PcTLR9B might play an important role in large yellow croaker defense against pathogenic infection.