Sphingomyelin Activates Hepatitis C Virus RNA Polymerase in a Genotype-Specific Manner

Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.Hepatitis C virus (HCV) has a positive-stranded RNA genome and belongs to the family Flaviviridae (21). HCV chronically infects more than 130 million people worldwide (34), and HCV infection often induces liver cirrhosis and hepatocellular carcinoma (19, 28). To date, pegylated interferon (PEG-IFN) and ribavirin are the standard treatments for HCV infection. However, many patients cannot tolerate their serious side effects. Therefore, the development of new and safer therapeutic methods with better efficacy is urgently needed.Lipids play important roles in HCV infection and replication. For example, the HCV core associates with lipid droplets and recruits nonstructural proteins and replication complexes to lipid droplet-associated membranes which are involved in the production of infectious virus particles (24). HCV RNA replication depends on viral protein association with raft membranes (2, 30). The association of cholesterol and sphingolipid with HCV particles is also important for virion maturation and infectivity (3). The inhibitors of the sphingolipid biosynthetic pathway, ISP-1 and HPA-12, which specifically inhibit serine palmitoyltransferase (SPT) (23) and ceramide trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus (37), suppress HCV virus production in cell culture but not viral RNA replication by the JFH1 replicon (3). Other serine SPT inhibitors (myriocin and NA255) inhibit genotype 1b replication (4, 29, 33). Very-low-density lipoprotein (VLDL) also interacts with the HCV virion (15).Sakamoto et al. reported that sphingomyelin bound to HCV RNA-dependent polymerase (RdRp) at the sphingomyelin binding domain (SBD; amino acids 230 to 263 of RdRp) to recruit HCV RdRp on the lipid rafts, where the HCV complex assembles, and that NA255 suppressed HCV replication by releasing HCV RdRp from the lipid rafts (29). In the present study, we analyzed the effect of sphingomyelin on HCV RdRp activity in vitro and found that sphingomyelin activated HCV RdRp activity in a genotype-specific manner. We also determined the sphingomyelin activation domain and the activation mechanism. Finally, we confirmed our biochemical data by a HCV replicon system.     

Single intra-arterial injection of mesenchymal stromal cells for treatment of steroid-refractory acute graft-versus-host disease: a pilot study

Background aimsCell therapy with mesenchymal stromal cells (MSC) has been reported recently as a promising treatment for severe acute graft-versus-host disease (GvHD).MethodsWe designed a pilot study to treat severe hepatic or gut GvHD using MSC derived from only the donor and cultured without bovine serum. Because the number of cultured MSC is smaller using this method, we planned to treat patients by intra-arterial regional administration directly to the target organs.ResultsThree patients were enrolled, and the MSC could be expanded using donor serum. There were no obvious side-effects immediately after arterial injection. The maximum response was partial in one of three patients and did not continue for more than 2 months. Idiopathic pneumonia syndrome developed in two of the three patients.ConclusionsA single local arterial MSC injection was unable to save these patients' lives and so might not be more effective than multiple systemic intravenous MSC injection. Further clinical research and additional strategies are required to develop appropriate methods for using MSC to achieve extended remission of GvHD.     

Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction

Long-chain fatty acids (FAs) are the predominant energy substrate utilized by the adult heart. The heart can utilize unesterified FA bound to albumin or FA obtained from lipolysis of lipoprotein-bound triglyceride (TG). We used heart-specific lipoprotein lipase knock-out mice (hLpL0) to test whether these two sources of FA are interchangeable and necessary for optimal heart function. Hearts unable to obtain FA from lipoprotein TG were able to compensate by increasing glucose uptake, glycolysis, and glucose oxidation. HLpL0 hearts had decreased expression of pyruvate dehydrogenase kinase 4 and increased cardiomyocyte expression of glucose transporter 4. Conversely, FA oxidation rates were reduced in isolated perfused hLpL0 hearts. Following abdominal aortic constriction expression levels of genes regulating FA and glucose metabolism were acutely up-regulated in control and hLpL0 mice, yet all hLpL0 mice died within 48 h of abdominal aortic constriction. Older hLpL0 mice developed cardiac dysfunction characterized by decreased fractional shortening and interstitial and perivascular fibrosis. HLpL0 hearts had increased expression of several genes associated with transforming growth factor-beta signaling. Thus, long term reduction of lipoprotein FA uptake is associated with impaired cardiac function despite a compensatory increase in glucose utilization.     

Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system

The microbial diversity and community succession of a circulation flush toilet were investigated by terminal restriction fragment length polymorphism and cloning analyses. Clonal libraries of 16S rRNA gene on day 3 and day 127 were constructed. On day 3, 102 clones were sequenced; Proteobacteria and Bacteroidetes accounted for 27% and 45%, respectively. On day 127, Proteobacteria had increased to 43% and Bacteroidetes had decreased to 26% of a total of 100 clones. Terminal restriction fragment length polymorphism peaks were identified by in silico analysis of clone libraries. The relative abundances of Nitrosomonas increased from 1% to 6% with commencement of nitrification and denitrification. Similarly, the relative abundance of terminal restriction fragments generated from Xanthomonas increased from 3% to 10%. Therefore, these bacteria could play a prominent role in this process. To reveal the relationship between stability of the microbial community and performance of the system, microbial community succession was visualized by multidimensional scaling analysis. The microbial community structure changed markedly, particularly during the start-up period of the system. The plots then became stable after the start of nitrification and denitrification. This result suggests that the succession of microbial community structure had a correlation with the performance of the system.     

Angiotensin II induces monocyte chemoattractant protein-1 expression via a nuclear factor-kappaB-dependent pathway in rat preadipocytes

Both monocyte chemoattractant protein-1 (MCP-1), a member of chemokine family, and angiotensinogen, a precursor of angiotensin (ANG) II, are produced by adipose tissue and increased in obese state. MCP-1 has been shown to decrease insulin-stimulated glucose uptake and several adipogenic genes expression in adipocytes in vitro, suggesting its pathophysiological significance in obesity. However, the pathophysiological interaction between MCP-1 and ANG II in adipose tissue remains unknown. The present study was undertaken to investigate the potential mechanisms by which ANG II affects MCP-1 gene expression in rat primary cultured preadipocytes and adipose tissue in vivo. ANG II significantly increased steady-state MCP-1 mRNA levels in a time- and dose-dependent manner. The ANG II-induced MCP-1 mRNA and protein expression was completely abolished by ANG II type 1 (AT1)-receptor antagonist (valsartan). An antioxidant/NF-kappaB inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1kappaB-alpha phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA expression. ANG II induced translocation of NF-kappaB p65 subunit from cytoplasm to nucleus by immunocytochemical study. Luciferase assay using reporter constructs containing MCP-1 promoter region revealed that two NF-kappaB binding sites in its enhancer region were essential for the ANG II-induced promoter activities. Furthermore, basal mRNA and protein of MCP-1 during preadipocyte differentiation were significantly greater in preadipocytes than in differentiated adipocytes, whose effect was more pronounced in the presence of ANG II. Exogenous administration of ANG II to rats led to increased MCP-1 expression in epididymal, subcutaneous, and mesenteric adipose tissue. In conclusion, our present study demonstrates that ANG II increases MCP-1 gene expression via ANG II type 1 receptor-mediated and NF-kappaB-dependent pathway in rat preadipocytes as well as adipose MCP-1 expression in vivo. Thus the augmented MCP-1 expression by ANG II in preadipocytes may provide a new link between obesity and cardiovascular disease.     

Anti-fibrotic effects of theophylline on lung fibroblasts

Theophylline has been used in the management of bronchial asthma and chronic obstructive pulmonary disease for over 50 years. It has not only a bronchodilating effect, but also an anti-inflammatory one conducive to the inhibition of airway remodeling, including subepithelial fibrosis. To date however, whether theophylline has a direct inhibitory effect on airway fibrosis has not been established. To clarify this question, we examined whether theophylline affected the function of lung fibroblasts. Theophylline suppressed TGF-beta-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited fibroblast proliferation stimulated by FBS and TGF-beta-induced alpha-SMA protein. A cAMP analog also inhibited TGF-beta-induced COL1 mRNA expression in lung fibroblasts. A PKA inhibitor reduced the inhibitory effect of theophylline on TGF-beta-induced COL1 mRNA expression. These results indicate that theophylline exerts anti-fibrotic effects, at least partly, through the cAMP-PKA pathway.     

Activation of diacylglycerol O-acyltransferase 1 gene results in increased tumor necrosis factor-alpha gene expression in 3T3-L1 adipocytes

The tumor necrosis factor-alpha (TNF-alpha) expression has been reported to be largely dependent on the size of adipocytes. We herein investigated the gene regulation of diacylglycerol O-acyltransferase (DGAT) in order to clarify the mechanism of TNF-alpha expression induced in large adipocytes. 3T3-L1 cells were cultured in the presence of 5 mM or 25 mM glucose to generate adipocytes from which the triglyceride content differs. The expression of TNF-alpha, DGAT1, and DGAT2 were upregulated in adipocytes cultured with 25 mM glucose. Furthermore, knockdown of DGAT1 gene significantly inhibited the TNF-alpha expression. Finally, the DGAT1 expression levels were closely related to the TNF-alpha level in 3T3-L1 adipocytes.     

Characterization of mouse tissue kallikrein 5

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (Klk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.     

In vivo generation of free radicals in the skin of live mice under ultraviolet light, measured by L-band EPR spectroscopy

Although free radicals may be involved in various types of UV-induced injuries, only a few in vivo studies of the generation of free radicals, including oxygen radicals, during exposure to ultraviolet light (UV) have been reported. In this study, the nitroxyl probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was intravenously injected into hairless mice, and its decay was monitored in the skin with an in vivo EPR spectrometer equipped with a surface-coil-type resonator. The rate of decay of the EPR signal increased during UV (UVA+B) irradiation. This increase in signal decay was suppressed by preadministration of a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN). PBN did not change the rate of signal decay in nonirradiated mice. The correlation between signal decay rate and physiological parameters such as blood velocity, blood mass, or skin temperature was low. The decay rate responded rapidly and reversibly to starting and stopping the UV illumination. Hydroxyl and peroxyl radicals caused reduction of the probe signal in vitro, and PBN inhibited only the peroxyl radical-induced signal reduction. These observations suggest that peroxyl radicals are generated in the skin of live mice during UVA+B irradiation.     

Suppression of AGE precursor formation following unilateral ureteral obstruction in mouse kidneys by transgenic expression of alpha-dicarbonyl/L-xylulose reductase

Unilateral ureteral obstruction (UUO) of kidneys causes acute generation of carbonyl stress. By electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS) we measured the content of methyl glyoxal, glyoxal, and 3-deoxyglucosone in mouse kidney extracts following UUO. UUO resulted in elevation of these dicarbonyls in the obstructed kidneys. Furthermore, the accumulation of 3-deoxyglucosone was significantly reduced in the kidneys of mice transgenic for alpha-dicarbonyl/L-xylulose reductase (DCXR) as compared to their wild-type littermates, demonstrating 4.91+/-2.04 vs. 6.45+/-1.85 ng/mg protein (P=0.044) for the obstructed kidneys, and 3.68+/-1.95 vs. 5.20+/-1.39 ng/mg protein (P=0.026) for the contralateral kidneys. On the other hand, collagen III content in kidneys showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal alpha-dicarbonyl compounds under oxidative circumstances, but it was not sufficient to suppress acute renal fibrosis during 7 d of UUO by itself.