Characteristics of settling matter and its role in nutrient cycles in a deep oligotrophic lake

The settling flux of seston (dry weight, DW), chlorophyll a (Chl a), particulate organic carbon (POC), particulate organic nitrogen (PON), and particulate phosphorus (PP) was measured monthly in 1981–1983 at 10 different depths in Lake Chuzenji, Japan; an oligotrophic lake with a maximum depth of 163 m. The Ti concentration in entrapped matter was used to separate the sedimentation flux into allochthonous and autochthonous components. Inflow loads of dissolved nutrients (DN: 4.5, DP: 0.48 g m^-2a^-1) were almost sufficient to supply the autochthonous fluxes at 30 m (PON: 2.9, PP: 0.51 g m^-2a^-1 ), and this flux of POC (26.6 g m^-2a ^-1) was about one-third of primary production (84 g C M^-2a^-1). Sedimentation of particulate matter was the main path of losing nutrients from lake water, explaining more than 80% removal of inflow loads (TN, TP). Decomposition rates during sedimentation which were calculated from the vertical difference in the autochthonous flux agreed very closely with the results obtained by laboratory experiments of a 100-day incubation (content ratios from field observations were: POC 0.67, PON 0.65, PP 0.85; and from laboratory experiments they were: POC 0.68, PON 0.70, PP 0.94). These decomposition rates and those near the sediment interface were used to explain dissolved oxygen depletion and nitrate increase in the hypolimnion during stratification. The average sinking velocities were 1.82m d^-1 for seston and 1.16 m d^-1 for Chl a at 30m, they were influenced by Chl a content of seston.     

Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release

Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl beta-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4 degrees C but shifted to lipid rafts at 37 degrees C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.     

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.     

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.     

Berberine alters the processing of Alzheimer's amyloid precursor protein to decrease Abeta secretion

Berberine is an isoquinoline alkaloid isolated from Coptidis rhizoma, a major herb widely used in Chinese herbal medicine. Berberine's biological activity includes antidiarrheal, antimicrobial, and anti-inflammatory effects. Recent findings show that berberine prevents neuronal damage due to ischemia or oxidative stress and that it might act as a novel cholesterol-lowering compound. The accumulation of amyloid-beta peptide (Abeta) derived from amyloid precursor protein (APP) is a triggering event leading to the pathological cascade of Alzheimer's disease (AD); therefore the inhibition of Abeta production should be a rational therapeutic strategy in the prevention and treatment of AD. Here, we report that berberine reduces Abeta levels by modulating APP processing in human neuroglioma H4 cells stably expressing Swedish-type of APP at the range of berberine concentration without cellular toxicity. Our results indicate that berberine would be a promising candidate for the treatment of AD.     

Hepatitis C virus RNA replication in human stellate cells regulates gene expression of extracellular matrix-related molecules

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, including chronic hepatitis, fibrosis, and cirrhosis. Fibrosis often develops in HCV-infected livers and ultimately leads to cirrhosis and carcinoma. During fibrosis, hepatic stellate cells (HSC) play important roles in the control of extracellular matrix synthesis and degradation in fibrotic livers. In this study, we established a subgenomic replicon (SGR) cell line with human hepatic stellate cells to investigate the effect of HCV RNA replication on HSC. Isolated SGR clones contained HCV RNA copy numbers ranging from 10⁴ to 10⁷ per μg total RNA, and long-term culture of low-copy number SGR clones resulted in markedly increased HCV RNA copy numbers. Furthermore, HCV RNA replication affected gene expression of extracellular matrix-related molecules in both hepatic stellate cells and hepatic cells, suggesting that HCV RNA replication and/or HCV proteins directly contribute to liver fibrosis. The HCV RNA-replicating hepatic stellate cell line isolated in this study will be useful for investigating hepatic stellate cell functions and HCV replication machinery.     

Involvement of MAP3K8 and miR-17-5p in Poor Virologic Response to Interferon-Based Combination Therapy for Chronic Hepatitis C

Despite advances in chronic hepatitis C treatment, a proportion of patients respond poorly to treatment. This study aimed to explore hepatic mRNA and microRNA signatures involved in hepatitis C treatment resistance. Global hepatic mRNA and microRNA expression profiles were compared using microarray data between treatment responses. Quantitative real-time polymerase chain reaction validated the gene signatures from 130 patients who were infected with hepatitis C virus genotype 1b and treated with pegylated interferon-alpha and ribavirin combination therapy. The correlation between mRNA and microRNA was evaluated using in silico analysis and in vitro siRNA and microRNA inhibition/overexpression experiments. Multivariate regression analysis identified that the independent variables IL28B SNP rs8099917, hsa-miR-122-5p, hsa-miR-17-5p, and MAP3K8 were significantly associated with a poor virologic response. MAP3K8 and miR-17-5p expression were inversely correlated with treatment response. Furthermore, miR-17-5p repressed HCV production by targeting MAP3K8. Collectively, the data suggest that several molecules and the inverse correlation between mRNA and microRNA contributed to a host genetic refractory hepatitis C treatment response.