Search

Phosphosites increased >50% upon treatment of KO cells with either CX4945 (A) or GO286 (B). Phosphosites increased more than 50% in CK2α(-/-)/Δα’ cells after CX4945 treatment (A), GO286 treatment (B). Phosphosites present in both are shown in the bottom part of the table labeled as “CX4945 & GO289” (C). Red color denotes phosphosites that do not conform to the CK2 consensus.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats, but relatively harmless to other rodents and mammals. As a vasoactive agent, NRB induces a species-specific vasocontractile effect that is restricted to the peripheral arteries of the rat. Despite the precise mechanisms behind this phenomenon having yet to be fully clarified, it is postulated that the molecular target of NRB could be located within the plasma membrane of rat peripheral artery myocytes (e.g. rat caudal artery myocytes). As such, the primary objective of this study was to develop a fluorescently labelled derivative of NRB to investigate its subcellular distribution/localization in both NRB-sensitive (freshly isolated rat caudal artery myocytes, FIRCAMs) and NRB-insensitive (human hepatic stellate, LX2) cells. Of the examples prepared, lead structure endo-NRB-NBD-bPA subsequently demonstrated retention of the parent toxicant's pharmacological profile (in terms of its ability to induce both a vasocontractile response in rat caudal artery rings in vitro, and a lethal end-point in rats in vivo). Endo-NRB-NBD-bPA was also shown to be significantly less permeable (an integral feature in the design of fluorescent probes targeting cell-surface receptors) to both LX2 cells and FIRCAMs. Disappointingly, no fluorescence could be observed on the plasma membrane of FIRCAMs stained with endo-NRB-NBD-bPA.

Farnesoid-X-receptor (FXR) and GP-BAR1 are bile acids receptors mainly expressed in entero-hepatic tissues that regulates some metabolic and non-metabolic functions. In the last ten years, this two receptors gained an increasing importance because they are involved in many physiological and physio-pathological human conditions. One of the main problem of these receptors is that bile acids are promiscuous ligand for both receptors. Even if the dual activation could be a promising pharmacological opportunity for several metabolic deseases, often it is associated to several side effects. Starting from the results obtained in several our previous works, in this contest, we decide to manipulate bile acids scaffold in order to produce new selective FXR and GP-BAR1 modulators.

BackgroundThe human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life's cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.AimIn this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.MethodsLX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.ResultsExposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.ConclusionsThe HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.

Background Persistent residual immune activation and lipid dysmetabolism are characteristics of HIV positive patients receiving an highly active antiretroviral therapy (HAART). Nuclear Receptors are transcription factors involved in the regulation of immune and metabolic functions through the modulation of gene transcription. The objective of the present study was to investigate for the relative abundance of members of the nuclear receptor family in monocytic cells isolated from HIV positive patients treated or not treated with HAART. Methods Monocytes isolated from peripheral blood mononuclear cells (PBMC) were used for analysis of the relative mRNA expressions of FXR, PXR, LXR, VDR, RARα, RXR, PPARα, PPARβ, PPARγ and GR by Real-Time polymerase chain reaction (PCR). The expression of a selected subset of inflammatory and metabolic genes MCP-1, ICAM-1, CD36 and ABCA1 was also measured. Results Monocytes isolated from HIV infected patients expressed an altered pattern of nuclear receptors characterized by a profound reduction in the expressions of FXR, PXR, PPARα, GR, RARα and RXR. Of interest, the deregulated expression of nuclear receptors was not restored under HAART and was linked to an altered expression of genes which supports both an immune activation and altered lipid metabolism in monocytes. Conclusions Altered expression of genes mediating reciprocal regulation of lipid metabolism and immune function in monocytes occurs in HIV. The present findings provide a mechanistic explanation for immune activation and lipid dysmetabolism occurring in HIV infected patients and could lead to the identification of novel potential therapeutic targets.