7,8-Dihydroxyflavone is a recently identified small molecular tropomyosin-receptor-kinase B (TrkB) agonist. Our preliminary structural activity relationship (SAR) study showed that the 7,8-dihydroxy groups are essential for the agonistic effect. To improve the lead compound's agonistic activity, we have conducted an extensive SAR study and synthesized numerous derivatives. We have successfully identified 4'-dimethylamino-7,8-dihydroxyflavone that displays higher TrkB agonistic activity than the lead. This novel compound also exhibits a more robust and longer TrkB activation effect in animals. Consequently, this new compound reveals more potent anti-apoptotic activity. Interestingly, chronic oral administration of 4'-dimethylamino-7,8-dihydroxyflavone and its lead strongly promotes neurogenesis in dentate gyrus and demonstrates marked antidepressant effects. Hence, our data support that the synthetic 4'-dimethylamino-7,8-dihydroxyflavone and its lead both are orally bioavailable TrkB agonists and possess potent antidepressant effects.

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Asparaginyl endopeptidase (AEP or legumain) is a lysosomal cysteine protease that cleaves protein substrates on the C-terminal side of asparagine. AEP plays a pivotal role in the endosome/lysosomal degradation system and is implicated in antigen processing. The processing of the lysosomal proteases cathepsins in kidney is completely defective in AEP-deficient mice with accumulation of macromolecules in the lysosomes, which is typically seen in lysosomal disorders. Here we show that mutant mice lacking AEP develop fever, cytopenia, hepatosplenomegaly, and hemophagocytosis, which are primary pathological manifestations of hemophagocytic syndrome/hemophagocytic lymphohistiocytosis (HLH). Moreover, AEP deficiency provokes extramedullary hematopoiesis in the spleen and abnormally enlarged histiocytes with ingested red blood cells (RBCs) in bone marrow. Interestingly, RBCs from AEP-null mice are defective in plasma membrane components. Further, AEP-null mice display lower natural killer cell activity, but none of the major cytokines is substantially abnormal. These results indicate that AEP might be a previously unrecognized component in HLH pathophysiology.

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Recent studies have indicated that ghrelin stimulates growth hormone release from the pituitary via the growth hormone secretagogue receptor (GHSR). We have previously isolated two GHSR subtypes from the pituitary of the black seabream Acanthopagrus schlegeli. In the present study, we have cloned and characterized ghrelin from the same fish species at both the cDNA and gene levels. The full-length seabream ghrelin cDNA, isolated from seabream stomach using a novel approach by exploiting a single conserved region in the coding region, was found to encode a prepropeptide of 107 amino acids, with the predicted mature ghrelin peptide consisting of 20 amino acids (GSSFLSPSQKPQNRGKSSRV). Embedded in this full-length cDNA is a putative fish orthologue of the recently reported mammalian obestatin peptide. The ghrelin gene in black seabream, obtained by genomic PCR, was found to encompass four exons and three introns, possessing the same structural organization as in tilapia and goldfish, but different from that in rainbow trout. In addition, a 2230-bp 5 -flanking region of the seabream ghrelin gene was obtained by genome walking. Sequence analysis revealed that, as in the case of the human ghrelin gene, there is neither a GC box nor a CAAT box present in the isolated 5 -flanking region. However, a number of putative transcription factorbinding sites different from the human counterpart were found in the 5 -flanking region of the seabream ghrelin gene, suggesting that different cis-and trans-acting elements are involved in controlling their gene expression. Functional activity of this 5 -flanking region was examined by cloning it into the pGL3-Basic vector upstream of the luciferase reporter gene and transfected into various cell lines. Positive promoter activity could only be recorded in the colon-derived Caco-2 cells, suggesting that the cloned 5 -flanking region represents the functional promoter of the seabream ghrelin gene, which exhibits tissue-specific promoter activity. Using reverse transcriptase PCR analysis, expression of ghrelin was detected only in the seabream stomach, but not in the other tissues examined, including the brain, gill, intestine, kidney, liver and spleen. This stomach-specific expression of ghrelin in seabream is subject to regulation, as administration of growth hormone or ipamorelin to the fish in vivo was demonstrated to enhance its expression. Reminiscent of the homologous upregulation found in the transcriptional control of the seabream GHSR gene, a similar homologous regulatory mechanism might also exist in controlling the expression of seabream ghrelin. The identification of both GHSR and ghrelin from a single fish species would facilitate our subsequent studies on the elucidation of the physiological functions of the ghrelin/GHSR system in teleost. The possible existence of obestatin in teleost opens up new research avenues on the somatotropic axis in fish.

Ebp1, an ErbB3 receptor-binding protein, inhibits cell proliferation and acts as a putative tumor suppressor. Ebp1 translocates into the nucleus and functions as a transcription corepressor for E2F-1. Here, we show that Ebp1 p42 isoform can be sumoylated on both K93 and K298 residues, which mediate its nuclear translocation and is required for its anti-proliferative activity. We find that TLS/FUS, an RNA-binding nuclear protein that is involved in pre- mRNA processing and nucleocytoplasmic shuttling, has Sumo1 E3 ligase activity for Ebp1 p42. Ebp1 directly binds TLS/FUS, which is regulated by genotoxic stress-triggered phosphorylation on Ebp1. Ebp1 sumoylation facilitates its nucleolar distribution and protein stability. Overexpression of TLS enhances Ebp1 sumoylation, while depletion of TLS abolishes Ebp1 sumoylation. Moreover, Unsumoylated Ebp1 mutants fail to suppress E2F-1- regulated transcription, resulting in loss of its anti-proliferation activity. Hence, TLS-mediated sumoylation is required for Ebp1 transcription repressive activity.