Oxidative stress and pro-apoptotic conditions in a rodent model of Wilson's disease

Wilson's disease (WD) is an inherited disorder, characterized by selective copper deposition in liver and brain, chronic hepatitis and extra-pyramidal signs. In this study, we investigated changes of biochemical markers of oxidative stress and apoptosis in liver, striatum and cerebral cortex homogenates from Long-Evans Cinnamon (LEC) rats, a mutant strain isolated from Long Evans (LE) rats, in whom spontaneous hepatitis develops shortly after birth. LEC and control (LE) rats at 11 and 14 weeks of age were used. We determined tissue levels of glutathione (GSH/GSSG ratio), lipid peroxides, protein-thiols (P-SH), nitric oxide metabolites, activities of caspase-3 and total superoxide-dismutase (SOD), striatal levels of monoamines and serum levels of hepatic amino-transferases. We observed a decrease of protein-thiols, GSH/GSSG ratio and nitrogen species associated to increased lipid peroxidation in the liver and striatum - but not in the cerebral cortex - of LEC rats, accompanied by dramatic increase in serum amino-transferases and decrease of striatal catecholamines. Conversely, SOD and caspase-3 activity increased consistently only in the cortex of LEC rats. Hence, we assume that enhanced oxidative stress may play a central role in the cell degeneration in WD, at the main sites of copper deposition, with discrete pro-apoptotic conditions developing in distal areas.     

Impact of adiponectin gene polymorphisms on plasma lipoprotein and adiponectin concentrations of viscerally obese men

The aim of this study was first to examine the relationships between adiponectin gene (Apm1) polymorphisms and anthropometric indices as well as plasma adiponectin and lipoprotein/lipid levels, and then to investigate whether the presence of visceral obesity or insulin resistance may modulate the impact of these polymorphisms on metabolic risk variables. Molecular screening of the Apm1 gene was achieved, and a sample of 270 unrelated men recruited from the greater Quebec City area and selected to cover a wide range of body fatness values was genotyped. Sequencing of the Apm1 gene revealed two previously reported polymorphisms (c.45T>G and c.276G>T) as well as two newly identified genetic variations (-13752delT and -13702G>C). Carriers of the c.276T allele had higher LDL-cholesterol and lower HDL-triglyceride concentrations than did 276G/G homozygotes (P=0.02 and P=0.01, respectively). Carriers of the c.45G allele exhibited higher plasma adiponectin concentrations than did 45T/T homozygotes (P=0.04). After dividing each genotype group into subgroups for visceral AT, homozygotes for the normal allele at position -13752delT, carriers of the c.45G allele, and carriers of the c.276T allele had similar total apolipoprotein B (apoB) concentrations, whether they were viscerally obese or not. These results suggest that some Apm1 gene polymorphisms influence plasma adiponectin concentrations and lipoprotein/lipid levels. In addition, the impact of these polymorphisms is modulated by the presence of visceral obesity.     

The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes: implications for viral infectivity

The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells: an intracellular, membrane-associated form essential for viral replication, a cell surface-associated form that may be involved in signal transduction, and a secreted form (sNS1), the biological properties of which remain elusive. To determine the distribution of the dengue virus (DEN) sNS1 protein in vivo, we have analyzed by immunohistological means the tissue tropism of purified DEN sNS1 injected intravenously into adult mice. The sNS1 protein was found predominantly associated with the liver, where hepatocytes appeared to represent a major target cell. We further showed that sNS1 could be efficiently endocytosed by human Huh7 and HepG2 hepatocytes in vitro. After its internalization, the protein was detected intracellularly for at least 48 h without being substantially degraded. Colocalization studies of sNS1 with markers of the endolysosomal compartments revealed that the protein was specifically targeted to lysobisphosphatidic acid-rich structures reminiscent of late endosomes, as confirmed by electron microscopy. Intracellular accumulation of sNS1 in Huh7 cells enhanced the fluid phase uptake of rhodamine-labeled dextran. Furthermore, preincubation of Huh7 cells with sNS1 increased dengue virus production after infection with the homologous strain of DEN-1 virus. Our results demonstrate that the accumulation of DEN sNS1 in the late endosomal compartment of hepatocytes potentializes subsequent dengue virus infection in vitro, raising the possibility that sNS1 may contribute to viral propagation in vivo.     

Short chain fatty acid and glucose metabolism in isolated pig colonocytes: modulation by NH4 +

Using monolayer cultures of costal chondrocytes established from four week old Clun Forest lambs, we have demonstrated that, under serum free conditions the cells release three IGFBPs (32, 29 and 21 kDa) into the medium. The most abundant of these—the 32 kDa BP-was shown to be IGFBP-2 by Western blotting. Furthermore we demonstrate that the levels of IGFBP 2 in conditioned medium are acutely increased (6, 12 and 24 h time points) following treatment of cells with bovine GH (1–100 ng/ml).In a parallel set of experiments, using ovine fibroblasts (derived from dermis) we show that IGFBPs of Mr 32, 29 and 21 kDa are also secreted by this cell type. However the relative abundance of these BPs differed from that seen in the chondrocyte cultures, with the 21 kDa species now the most abundant. In addition, prolonged exposure of autoradiographs indicated that fibroblasts secreted a higher Mr IGFBP (most probably ovine BP-3) that was not detected in any of our chondrocyte cultures. Most significant however was the demonstration that bGH did not dramatically affect the levels of IGFBPs in fibroblast cell cultures. We conclude that GH stimulates BP-2 production from chondrocytes and this is a cell-type specific effect in as much as it is not replicated in cultures of dermal fibroblasts.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CM conditioned medium - DMEM Dulbecco's Modified Eagles Medium - FCS foetal calf serum - IGF insulin-like growth factor - IGFBP insulin-like growth factor binding protein - HBSS Hanks' Balanced Salt Solution - GH growth hormone - NBT nitroblue tetrazolium - SFM serum free medium - TBS tris buffered saline     

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.     

Interferon-γ Is a Crucial Activator of Early Host Immune Defense against Mycobacterium ulcerans Infection in Mice

Buruli ulcer (BU), caused by infection with Mycobacterium ulcerans, is a chronic necrotizing human skin disease associated with the production of the cytotoxic macrolide exotoxin mycolactone. Despite extensive research, the type of immune responses elicited against this pathogen and the effector functions conferring protection against BU are not yet fully understood. While histopathological analyses of advanced BU lesions have demonstrated a mainly extracellular localization of the toxin producing acid fast bacilli, there is growing evidence for an early intra-macrophage growth phase of M. ulcerans. This has led us to investigate whether interferon-γ might play an important role in containing M. ulcerans infections. In an experimental Buruli ulcer mouse model we found that interferon-γ is indeed a critical regulator of early host immune defense against M. ulcerans infections. Interferon-γ knockout mice displayed a faster progression of the infection compared to wild-type mice. This accelerated progression was reflected in faster and more extensive tissue necrosis and oedema formation, as well as in a significantly higher bacterial burden after five weeks of infection, indicating that mice lacking interferon-γ have a reduced capacity to kill intracellular bacilli during the early intra-macrophage growth phase of M. ulcerans. This data demonstrates a prominent role of interferon-γ in early defense against M. ulcerans infection and supports the view that concepts for vaccine development against tuberculosis may also be valid for BU.     

Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.