HBeAg induces the expression of macrophage miR-155 to accelerate liver injury via promoting production of inflammatory cytokines

Activation of Kupffer cells (KCs) induced that inflammatory cytokine production plays a central role in the pathogenesis of HBV infection. The previous studies from our and other laboratory demonstrated miRNAs can regulate TLR-inducing inflammatory responses to macrophage. However, the involvement of miRNAs in HBV-associated antigen-induced macrophage activation is still not thoroughly understood. Here, we evaluated the effects and mechanisms of miR-155 in HBV-associated antigen-induced macrophage activation. First, co-culture assay of HepG2 or HepG2.2.15 cells and RAW264.7 macrophages showed that HepG2.2.15 cells could significantly promote macrophages to produce inflammatory cytokines. Furthermore, we, respectively, stimulated RAW264.7 macrophages, mouse primary peritoneal macrophages, or healthy human peripheral blood monocytes with HBV-associated antigens, including HBcAg, HBeAg, and HBsAg, and found that only HBeAg could steadily enhance the production of inflammatory cytokines in these cells. Subsequently, miRNAs sequencing presented the up- or down-regulated expression of multiple miRNAs in HBeAg-stimulated RAW264.7 cells. In addition, we verified the expression of miR-155 and its precursors BIC gene with q-PCR in the system of co-culture or HBeAg-stimulated macrophages. Meanwhile, the increased miR-155 expression was positively correlation with serum ALT, AST, and HBeAg levels in AHB patients. Although MAPK, PI3K, and NF-κB signal pathways were all activated during HBeAg treatment, only PI3K and NF-κB pathways were involved in miR-155 expression induced by HBeAg stimulation. Consistently, miR-155 over-expression inhibited production of inflammatory cytokines, which could be reversed by knocking down miR-155. Moreover, we demonstrated that miR-155 regulated HBeAg-induced cytokine production by targeting BCL-6, SHIP-1, and SOCS-1. In conclusion, our data revealed that HBeAg augments the expression of miR-155 in macrophages via PI3K and NF-κB signal pathway and the increased miR-155 promotes HBeAg-induced inflammatory cytokine production by inhibiting the expression of BCL-6, SHIP-1, and SOCS-1.     

Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-β-lactamase

The CphA metallo--lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.Received 3 March 2003; received after revision 4 August 2003; accepted 25 August 2003     

Novel rearrangement of purines

Summary 6-Trichloromethyl-9-methylpurine (1) rearranges to 6-dichloromethyl-9-methyl-8-oxopurine (2) in aqueous mild acidic solution. The rearrangement is rationalized in terms of a reaction involving protonation, covalent hydration, prototropic equilibrium and/or a hydride transfer. An alternative mechanism involving a positive halogen compound and hypochlorous acid as an intermediary is also proposed. Compound1 condenses with 4,5-diaminopyrimidine to give the purine-pyrimidine Schiff base pair4.Acknowledgments. We are deeply indebted to Professor S. Cohen of the Sackler School of Medicine, Tel Aviv University (Ramat Aviv, Isreal) for his advice and encouragement. Support of this research by the Israel Cancer Association, the Ber-Lamsdorf Foundation Switzerland Israel and by the Advancement of Mankind Foundation, is gratefully acknowledged. We thank Proff. D. Arigoni and A. Eschenmoser, ETH Zürich, for their valuable proposals and comments on the mechanism of the rearrangement.     

Green tea epigallocatechin-3-gallate is an inhibitor of mammalian histidine decarboxylase

(–)-Epigallocatechin-3-gallate, an antiproliferative and antiangiogenic component of green tea, has been reported to inhibit dopa decarboxylase. In this report, we show that this compound also inhibits histidine decarboxylase, the enzymic activity responsible for histamine biosynthesis. This inhibition was proved by a double approach, activity measurements and UV-Vis spectra of enzyme-bound pyridoxal-5-phosphate. At 0.1mM (–)-epigallocatechin-3-gallate, histidine decarboxylase activity was inhibited by more than 60% and the typical spectrum of the internal aldimine form shifted to a stable major maximum at 345nm, suggesting that the compound causes a stable change in the structure of the holoenzyme. Since histamine release is one of the primary events in many inflammatory responses, a new potential application of (–)-epigallocatechin-3-gallate in prevention or treatment of inflammatory processes is suggested by these data.Received 8 April 2003; received after revision 20 May 2003; accepted 3 June 2003     

Evolutionary and functional epitopes of the Spätzle protein: New insights into activation of the Toll receptor

Sp?tzle, a dimeric ligand, binds to the Drosophila Toll receptor and activates the signal pathway functioning in both embryonic patterning and innate immunity. Here, we used the evolutionary trace approach based on phylogenetic information to predict the evolutionary epitope of Sp?tzle and found that it mainly clusters in several adjacent loops of Sp?tzle far from the cystine-knot structural domain. We designed six mutants of Sp?tzle based on the evolutionary epitope and transfected them into a stable cell line expressing the luciferase reporter gene under the control of the drosomycin promoter. Luciferase assays showed that these mutants cannot significantly activate the drosomycin promoter, suggesting the involvement of these sites in binding of Sp?tzle to the Toll receptor. These data highlight the importance of the Trp-loop of the mushroom-shaped Sp?tzle dimer in Toll receptor activation and demonstrate that evolution-guided site-specific mutagenesis represents a useful and promising strategy for understanding the ligand-receptor interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 14 January 2009; received after revision 14 February 2009; accepted 09 March 2009     

Apoptotic cell-derived factors induce arginase II expression in murine macrophages by activating ERK5/CREB

Apoptotic cell (AC)-derived factors alter the physiology of macrophages (MΦs) towards a regulatory phenotype, characterized by reduced nitric oxide (NO) production. Impaired NO formation in response to AC-conditioned medium (CM) was facilitated by arginase II (ARG II) expression, which competes with inducible NO synthase for l-arginine. Here we explored signaling pathways allowing CM to upregulate ARG II in RAW264.7 MΦs. Sphingosine-1-phosphate (S1P) was required and acted synergistically with a so far unidentified factor to elicit high ARG II expression. S1P activated S1P₂, since S1P₂ knockdown prevented ARG II upregulation. Furthermore, ERK5 knockdown attenuated CM-mediated ARG II protein induction. CREB was implicated as shown by EMSA analysis and decoy-oligonucleotides scavenging CREB in RAW264.7 MΦs, which blocked ARG II expression. We conclude that AC-derived S1P binds to S1P₂ and acts synergistically with other factors to activate ERK5 and concomitantly CREB. This signaling cascade shapes an anti-inflammatory MΦ phenotype by ARG II induction.     

Ethanol and opioid receptor signalling

Summary Ethanol may modulate endogenous opioid systems by disrupting opioid receptor signalling. Low concentrations of ethanol slightly potentiate -opioid receptor binding by increasing receptor B_max, and, in some cases, chronic ethanol exposure decreases the density or affinity of the -opioid receptors. By contrast, high concentrations of ethanol acutely decrease -opioid receptor binding by decreasing receptor affinity, whereas chronic exposure of animals and neuronal cell lines to lower concentrations of ethanol leads to possibly adaptive increases in the density or affinity of the -opioid receptors. In the neuronal cell line NG108-15, ethanol does not up-regulate the -opioid receptor by blocking receptor degradation or endocytosis, but protein synthesis is required for this response. Up-regulation of the -opioid receptor renders ethanol-treated NG108-15 cells 3.5-fold more sensitive to opioid inhibition of adenylyl cyclase. Long-term treatment with ethanol also increases maximal opioid inhibition in NG108-15 cells, possibly by decreasing levels of G_s and its mRNA. Ethanol differentially modulates signal transduction proteins in three additional neuronal cell lines, N18TG2, N4TG1, and N1E-115. Ethanol-treated N18TG2 cells show the least up-regulation of the -opioid receptor, little heterologous desensitization of adenylyl cyclase, and no changes in G_s or G_i. By contrast, ethanol-treated N1E-115 cells show the largest up-regulation of the -opioid receptor, the most heterologous desensitization of adenylyl cyclase, and concentration-dependent decreases in G_s and increases in G_i. Further analysis of these related neuronal cell lines may help to identify the molecular elements that endow some, but not all, neuronal cells with the capacity to adapt to ethanol.