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Objective: To observe the effects of Huannao Yicong Formula (, HYF) on learning and memory and it's regulating effect on γ-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer's disease., Methods: Sixty APP/PS1 transgenic mice, randomly allocated into 4 groups, the model group, the donepezil group (0.65 mg/kg), HYF low-dose group (HYF-L, 5.46 g/kg) and HYF high-dose group (HYF-H, 10.92 g/kg), 15 for each group. Another 15 C57BL/6J mice with the same age and same genetic background were allocated into the control group, proper dosage of drugs or distilled water were given by intragastric administration once daily for 12 weeks. After 12 weeks of administration, the learning and memory abilities of mice in each group was evaluated by the morris water maze test, amyloid precursor protein (APP), Aβ 1-40 and Aβ 1-42 levels in hippocampus were detected by enzyme-linked immunosorbent assay, γ-secretase was detected by dual luciferase assaying, the levels of APH-1a, hypoxia-inducible factor 1α (HIF-1α), cAMP response element-binding protein (CREB) and PEN-2 and their mRNA expression was measured by Western blot and real-time polymerase chain reaction., Results: HYF can ameliorate learning and memory deficits in APP/PS1 transgenic mice by decreasing the escape latency, improving the number of platform crossing and swimming speed (P<0.01, P<0.05). HYF can decrease the levels of APP, Aβ 1-40 , Aβ 1-42 and the activity of γ-secretase in hippocampus of Alzheimer's disease model mice. HYF can down-regulate the levels of CREB and PEN-2 and the expression of their mRNA., Conclusion: HYF can improve the learning and memory ability by inhibiting the activity of γ-secretase through the CREB/PEN-2 signaling pathway, and this may be one of the therapeutic mechanisms of HYF in Alzheimer's disease.

Galectins are a family of endogenous lectins with β-galactosides affinity, playing significant roles in the innate immunity of vertebrates and invertebrates. In this report, a new galectin-9 cDNA was identified and characterized in large yellow croaker Larimichthys crocea (designated as LcGal-9). The complete cDNA sequence of LcGal-9 was 1795 bp, with an open reading frame (ORF) of 1032 bp encoding 343 amino acids. The putative LcGal-9 protein contained two carbohydrate recognition domains (CRDs) connected by a linker peptide, with each carrying two conserved β-galactoside binding motifs H-NPR and WG-EE-, and it possessed neither a signal peptide nor a transmembrane domain. LcGal-9 protein shared 43-74% identity with galectin-9 sequences from other species. The qRT-PCR analysis revealed that LcGal-9 mRNA was constitutively expressed in all tissues examined, predominately expressed in liver, spleen, gill, kidney, head-kidney and intestine. Western blot analysis showed that LcGal-9 protein was highly expressed in liver, spleen, intestine, kidney, head-kidney, skin, gill, and heart, but not detected in muscle and plasma. LcGal-9 mRNA transcripts were induced by poly I:C in the liver (from 6 h to 48 h), spleen (at 12 h) and head-kidney (at 12 h and 24 h). In contrast, Vibrio parahaemolyticus caused a significant down-regulation in these three tissues, except for in spleen of 48 h and head-kidney of 3 h. Post-infection with Cryptocaryon irritans, the transcripts were dramatically up-regulated in gill, skin, spleen and head-kidney during initial infection period, while significant down-regulation afterward was also observed both in spleen and head-kidney. The recombinant LcGal-9 (named as rLcGal-9) purified from Escherichia coli BL21 (DE3) demonstrated hemagglutination against human, rabbit and L. crocea in a Ca(2+)-independent manner, which was inhibited by α-Lactose and LPS. The results of bacterial agglutination assays showed that rLcGal-9 was able to agglutinate Gram-negative bacteria V. alginolyticus and Aeromonas hydrophila in a Ca(2+)-independent manner. By immunohistochemistry assay, significant increases of LcGal-9 protein appeared in the spleen stimulated with poly I:C (for 12 h) and V. parahaemolyticus (for 48 h) compared with the control. Based on the collective data, LcGal-9 might play an important role in innate immune responses, especially defense against Gram-negative bacteria in L. crocea., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

BCA2, as an E3 ubiquitin ligase, is an important anti-virus immune factor in mammals. Up to date, there are not any related reports on BCA2 protein in fishes yet. In the present investigation, BCA2 in large yellow croaker Larimichthys crocea (named as LcBCA2) was identified and characterized. The full-length cDNA of LcBCA2 was 1571 bp, including an ORF of 888 bp encoding a polypeptide of 295 amino acids. The putative LcBCA2 protein contained a RING-H2 motif at C terminal. The LcBCA2 transcripts were broadly distributed in all detected tissues, with high expression in muscle, moderate in blood, skin, heart, liver and spleen, weak in other tissue as indicated by qPCR analysis. Significant increases were observed in skin, gill and spleen after infection of Cryptocaryon irritans, and in spleen and head-kidney after inactivated Vibrio. parahaemolyticus, LPS and Poly I:C stimulations. Tissue localization by in-situ hybridization showed that LcBCA2 mainly expressed in the spleen of the fish in the test group. Our findings showed that LcBCA2 inclined to sharply increase in immune organs, especially in head-kidney after bacterial and viral stimulations, while in locations (skin and gill) of parasites infections, suggesting that BCA2 may play an important role in fish defense against bacteria, virus and parasites infections, but the immune mechanisms is are different., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

TBK1 has been extensively studied in mammals because of its important roles as a molecular bridge, linking the TLRs (TLR3 and TLR4) and RLRs signals to activate transcriptional factors IRF3 and IRF7 for IFN-I production. However, the information on molecular and functional characteristics of TBK1 in teleosts is limited. In this study, the molecular characterization and immune response of TBK1 in Larimichthys crocea (named as LcTBK1) as well as its interaction with Nrdp1 were investigated. Sequence analysis demonstrated that LcTBK1 included four functional motifs, the N-terminal protein kinase domain and ATP-binding site, middle ULD and C-terminal coiled-coil domain. The tissue expression profiles indicated that LcTBK1 gene was constitutively expressed in the twelve tissues examined, with high expression in brain. Temporal expression analysis showed that LcTBK1 mRNA was obviously increased in the liver after injection of LPS, Poly I:C and inactive Vibrio parahaemolyticus, however, declined at some time points in spleen and head-kidney. Furthermore, we found that LcTBK1 can interact with LcNrdp1, an E3 ubiquitin ligase that involved in immune response to Cryptocaryon irritans infection in L. crocea. The qPCR showed that LcNrdp1 was also significantly up-regulated in liver, down-regualted at some time points in spleen and head-kidney after LPS, Poly I:C and inactive V. parahaemolyticus injection, although the expression patterns of the two genes after the three treatments were different in change magnitude and up-regulation timespan. These results suggested that LcTBK1 was involved in L. crocea defense against the pathogen infection and can be regulated by Nrdp1 in PPRs signaling pathway of fishes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Toll-like receptor 3 (TLR3) plays an important role in innate immune responses. In this report, the full-length cDNA sequence and genomic structure of Pseudosciaena crocea TLR3 (PcTLR3) were identified and characterized. The full-length cDNA of PcTLR3 was of 3384 bp, including a 5'-terminal untranslated region (UTR) of 65 bp, a 3'-terminal UTR of 589 bp and an open reading frame (ORF) of 2730 bp encoding a polypeptide of 909 amino acid residues. The full-length genome sequence of PcTLR3 was composed of 5721 nucleotides, including five exons and four introns. The putative PcTLR3 protein contained a signal peptide sequence, 16 leucine-rich repeat (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR3 in most tissues, with the predominant expression in liver, then intestine, and the weakest expression in blood cells. The expression of PcTLR3 after injection with poly inosinic:cytidylic (I:C) and Vibrio parahemolyticus was tested in spleen, blood cells and liver. The results indicated that PcTLR3 transcripts could be induced in the three tissues by injection with poly I:C. The highest expression was in the blood cells with 43.5 times (at 6h) greater expression than in the control (p<0.05). In addition, after V. parahemolyticus challenge, a moderate up-regulation and down-regulation of PcTLR3 was found in blood cells and liver, respectively. Our results suggested that PcTLR3 might play an important role in fish's defense against both viral and bacterial infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The interferon regulatory factor (IRF) family is known to be crucial in mediating the host defense against pathogen infection by binding to characteristic elements in promoters within interferon (IFN) genes and IFN-inducible genes. In this report, the full-length cDNA of IRF1 was cloned from the large yellow croaker, Pseudosciaena crocea. It was of 1667 bp, including a 5'-terminal untranslated region (UTR) of 142 bp, a 3'-terminal UTR of 674 bp and an open reading frame (ORF) of 861 bp encoding a polypeptide of 286 amino acids. The putative amino acid sequence contained a typical IRF domain at the N-terminal. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of IRF1 in most detected tissues, with the predominant expression in the gill and spleen and the weakest expression in the brain. The expression of IRF1 after challenged with LPS and poly I:C was tested in blood, spleen and liver, which showed that IRF1 changed obviously with the most significantly up-regulated expression was 37 times (at 6 h) after injection with poly I:C in the blood and 13 times (at 3 h) after injection with LPS in the liver compared with the control values (p < 0.01). These results indicated that as a crucial factor in regulating the IFN and IFN-inducible elements in mammals, IRF1 might play an important role in large yellow croaker defense against the pathogen infection., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The effects of some pathogen-associated molecular patterns (PAMPs) (laminarin, LPS and poly I:C) on total hemocyte counts (THC), phenoloxidase (PO) activity, superoxide anion production and lectin, prophenoloxidase, lysozyme, cytosolic manganese superoxide dismutase (C-MnSOD) and catalase (CAT) gene expression were studied. The results showed that the production or activity of most tested immune factors and the expression of most tested genes were up-regulated after stimulation with PAMPs, among which the highest value of lectin with 4.4 times as much as that of the control group appeared at 6 h in hemocytes, of CAT with 47 times as much as that of the control group appeared at 12 h in hepatopancreas, and with 2.7 times higher than that of the control group at 24 h of C-MnSOD in hepatopancreas after laminarin injection. The peak value of proPO, lysozyme and C-MnSOD appeared at 6 h in hepatopancreas, 24 h in hepatopancreas and 24 h in hemocytes after LPS injection, respectively. The highest expression level of lysozyme appeared at 12 h in hemocytes after poly I:C injection. However, significant decreases of PO activity in hemocytes and lectin expression in hepatopancreas were found after poly I:C injection, and a dramatic down-regulation of proPO expression from 3 h to 48 h was found in hemocytes after injection with laminarin, LPS and poly I:C. The results suggest that the shrimp immune response could be activated or inhibited by different PAMPs, and that the hepatopancreas also plays a key role by synthesizing immune factors.

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.

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.

Upon light-induced nuclear translocation, phytochrome (phy) sensory photoreceptors interact with, and induce rapid phosphorylation and consequent ubiquitin-mediated degradation of, transcription factors, called PIFs, thereby regulating target gene expression and plant development. Nevertheless, the biochemical mechanism of phy-induced PIF phosphorylation has remained ill-defined. Here we identify a family of nuclear protein kinases, designated Photoregulatory Protein Kinases (PPK1-4; formerly called MUT9-Like Kinases (MLKs)), that interact with PIF3 and phyB in a light-induced manner in vivo. Genetic analyses demonstrate that the PPKs are collectively necessary for the normal light-induced phosphorylation and degradation of PIF3. PPK1 directly phosphorylates PIF3 in vitro, with a phosphosite pattern that strongly mimics the light-induced pattern in vivo. These data establish that the PPKs are directly involved in catalysing the photoactivated-phy-induced phosphorylation of PIF3 in vivo, and thereby are critical components of a transcriptionally centred signalling hub that pleiotropically regulates plant growth and development in response to multiple signalling pathways.

SWI/SNF-type chromatin remodelers, such as BRAHMA (BRM), and H3K27 demethylases both have active roles in regulating gene expression at the chromatin level, but how they are recruited to specific genomic sites remains largely unknown. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), a plant-unique H3K27 demethylase, targets genomic loci containing a CTCTGYTY motif via its zinc-finger (ZnF) domains and facilitates the recruitment of BRM. Genome-wide analyses showed that REF6 colocalizes with BRM at many genomic sites with the CTCTGYTY motif. Loss of REF6 results in decreased BRM occupancy at BRM-REF6 co-targets. Furthermore, REF6 directly binds to the CTCTGYTY motif in vitro, and deletion of the motif from a target gene renders it inaccessible to REF6 in vivo. Finally, we show that, when its ZnF domains are deleted, REF6 loses its genomic targeting ability. Thus, our work identifies a new genomic targeting mechanism for an H3K27 demethylase and demonstrates its key role in recruiting the BRM chromatin remodeler.

After light-induced nuclear translocation, phytochrome photoreceptors interact with and induce rapid phosphorylation and degradation of basic helix-loop-helix transcription factors, such as PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), to regulate gene expression. Concomitantly, this interaction triggers feedback reduction of phytochrome B (phyB) levels. Light-induced phosphorylation of PIF3 is necessary for the degradation of both proteins. We report that this PIF3 phosphorylation induces, and is necessary for, recruitment of LRB [Light-Response Bric-a-Brack/Tramtrack/Broad (BTB)] E3 ubiquitin ligases to the PIF3-phyB complex. The recruited LRBs promote concurrent polyubiqutination and degradation of both PIF3 and phyB in vivo. These data reveal a linked signal-transmission and attenuation mechanism involving mutually assured destruction of the receptor and its immediate signaling partner.

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.

When brassinosteroid levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. Brassinosteroid promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here, using tandem affinity purification, we identified protein phosphatase 2A (PP2A) as a BZR1-interacting protein. Genetic analyses demonstrated a positive role for PP2A in brassinosteroid signalling and BZR1 dephosphorylation. Members of the B' regulatory subunits of PP2A directly interact with BZR1's putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function for PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the brassinosteroid-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.