Serum acid sphingomyelinase is upregulated in chronic hepatitis C infection and non alcoholic fatty liver disease

Sphingolipids constitute bioactive molecules with functional implications in homeostasis and pathogenesis of various diseases. However, the role of sphingolipids as possible disease biomarkers in chronic liver disease remains largely unexplored. In the present study we used mass spectrometry and spectrofluorometry methods in order to quantify various sphingolipid metabolites and also assess the activity of an important corresponding regulating enzyme in the serum of 72 healthy volunteers as compared to 69 patients with non-alcoholic fatty liver disease and 69 patients with chronic hepatitis C virus infection. Our results reveal a significant upregulation of acid sphingomyelinase in the serum of patients with chronic liver disease as compared to healthy individuals (p < 0.001). Especially in chronic hepatitis C infection acid sphingomyelinase activity correlated significantly with markers of hepatic injury (r = 0.312, p = 0.009) and showed a high discriminative power. Accumulation of various (dihydro-) ceramide species was identified in the serum of patients with non-alcoholic fatty liver disease (p < 0.001) and correlated significantly to cholesterol (r = 0.448, p < 0.001) but showed a significant accumulation in patients with normal cholesterol values as well (p < 0.001). Sphingosine, a further bioactive metabolite, was also upregulated in chronic liver disease (p < 0.001). However, no significant correlation to markers of hepatic injury was identified. Conclusion: Chronic hepatitis C virus infection and non-alcoholic fatty liver disease induce a significant upregulation of serum acid sphingomyelinase which appears as a novel biomarker in chronic hepatopathies. Further studies are required to elucidate the potential of the sphingolipid signaling pathway as putative therapeutic target in chronic liver disease.     

Synthesis of Lipid Mediators during UVB-Induced Inflammatory Hyperalgesia in Rats and Mice

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.     

Experimental observations on the regioselectivity of glycosylation of a 4,6-diol system in the β-d-mannopyranosyl unit of a N-glycan pentasaccharide core structure

The regioselectivity of glycosylation of a 4,6-diol system in the β-mannopyranosyl unit of a N-glycan pentasaccharide core structure is found to be strongly dependent on the structure of the glycosyl donor. While glycosylation with a 2-O-acetyl-d-mannopyranosyl trichloroacetimidate and with a d-mannopyranosyl (α1→3) 2-O-acetyl mannopyranosyl trichoroacetimidate regioselectively occurs at the primary OH-6 position, reaction with d-mannopyranosyl (α1→6) mannopyranosyl 2-O-benzoyl, 2-O-acetyl and 2-O-pivaloyl trichloroacetimidate results in approximately 1:1 mixture of regioisomers at primary OH-6 and secondary OH-4 positions.     

Regulation of ribonucleotide reductase in response to iron deficiency

Ribonucleotide reductase (RNR) is an essential enzyme required for DNA synthesis and repair. Although iron is necessary for class Ia RNR activity, little is known about the mechanisms that control RNR in response to iron deficiency. In this work, we demonstrate that yeast cells control RNR function during iron deficiency by redistributing the Rnr2-Rnr4 small subunit from the nucleus to the cytoplasm. Our data support a Mec1/Rad53-independent mechanism in which the iron-regulated Cth1/Cth2 mRNA-binding proteins specifically interact with the WTM1 mRNA in response to iron scarcity and promote its degradation. The resulting decrease in the nuclear-anchoring Wtm1 protein levels leads to the redistribution of the Rnr2-Rnr4 heterodimer to the cytoplasm, where it assembles as an active RNR complex and increases deoxyribonucleoside triphosphate levels. When iron is scarce, yeast selectively optimizes RNR function at the expense of other non-essential iron-dependent processes that are repressed, to allow DNA synthesis and repair.     

The solution structure of double helical arabino nucleic acids (ANA and 2��F-ANA): effect of arabinoses in duplex-hairpin interconversion

We report here the first structure of double helical arabino nucleic acid (ANA), the C2′-stereoisomer of RNA, and the 2′-fluoro-ANA analogue (2′F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2′-deoxy-2′F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities. One structure is a monomeric hairpin in which the stem is formed by base paired 2′F-ANA nucleotides and the loop by unpaired ANA nucleotides. The second structure is a bimolecular duplex, with all the nucleotides (2′F-ANA and ANA) forming Watson–Crick base pairs. The duplex structure is canonical B-form, with all arabinoses adopting a pure C2′-endo conformation. In the ANA:ANA segment, steric interactions involving the 2′-OH substituent provoke slight changes in the glycosidic angles and, therefore, in the ANA:ANA base pair geometry. These distortions are not present in the 2′F-ANA:2′F-ANA regions of the duplex, where the –OH substituent is replaced by a smaller fluorine atom. 2′F-ANA nucleotides adopt the C2′-endo sugar pucker and fit very well into the geometry of B-form duplex, allowing for favourable 2′F···H8 interactions. This interaction shares many features of pseudo-hydrogen bonds previously observed in 2′F-ANA:RNA hybrids and in single 2′F-ANA nucleotides.     

A Novel Liposome-Based Nanocarrier Loaded with an LPS-dsRNA Cocktail for Fish Innate Immune System Stimulation

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.