Molecular Mechanism Underlying the Suppression of CPB2 Expression Caused by Persistent Hepatitis C Virus RNA Replication

The mechanisms of hepatitis C virus (HCV)-associated hepatocarcinogenesis and disease progression are unclear. We previously observed that the expression level of carboxypeptidase B2 (CPB2) gene was remarkably suppressed by persistent HCV RNA replication in human hepatoma cell line Li23-derived cells. The results of the present study demonstrated that the CPB2 expression in patients with chronic hepatitis C was inversely correlated with several risk factors of hepatic fibrosis or steatosis, although ectopic CPB2 expression did not suppress the expression of fibrogenic or lipogenic genes. The suppressed CPB2 expression was restored by treatment with 5-azacytidine. To clarify the mechanism underlying this phenomenon, we analyzed the CPB2 promoter, and the results revealed that (1) hepatocyte nuclear factor 1 (HNF1), especially HNF1α, was essential for the CPB2 promoter, and (2) CPB2 promoter was not methylated by persistent HCV RNA replication. The expression levels of HNF1α and HNF1β were also not changed by persistent HCV RNA replication. These results suggest the existence of 5-azacytidine-inducible or -reducible unknown factor(s) that can control the CPB2 expression. To evaluate this idea we performed a microarray analysis, and several gene candidates corresponding to the suggested factor(s) were identified

Genetic Characterization of Hepatitis C Virus in Long-Term RNA Replication Using Li23 Cell Culture Systems

Background　 　 The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. 　 Methodology/Principal Findings　 　 Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5′-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0–9.0×10−3 and 2.7–4.0×10−3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. 　 Conclusions/Significance　 　 Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis

Identification of host genes showing differential expression profiles with cell-based long-term replication of hepatitis C virus RNA

　Persistent hepatitis C virus (HCV) infection frequently causes hepatocellular carcinoma. However, the mechanisms of HCV-associated hepatocarcinogenesis and disease progression are unclear. Although the human hepatoma cell line, HuH-7, has been widely used as the only cell culture system for robust HCV replication, we recently developed new human hepatoma Li23 cell line-derived OL, OL8, OL11, and OL14 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates. OL, OL8, OL11, and OL14 cells were cultured for more than 2 years. We prepared cured cells from OL8 and OL11 cells by interferon-γ treatment. The cured cells were also cultured for more than 2 years. cDNA microarray and RT-PCR analyses were performed using total RNAs prepared from these cells. We first selected several hundred highly or moderately expressed probes, the expression levels of which were upregulated or downregulated at ratios of more than 2 or less than 0.5 in each set of compared cells (e.g., parent OL8 cells versus OL8 cells cultured for 2 years). From among these probes, we next selected those whose expression levels commonly changed during a 2-year culture of genome-length HCV RNA-replicating cells, but which did not change during a 2-year culture period in cured cells. We further examined the expression levels of the selected candidate genes by RT-PCR analysis using additional specimens from the cells cultured for 3.5 years. Reproducibility of the RT-PCR analysis using specimens from recultured cells was also confirmed. Finally, we identified 5 upregulated genes and 4 downregulated genes, the expression levels of which were irreversibly altered during 3.5-year replication of HCV RNA. These genes may play roles in the optimization of the environment in HCV RNA replication, or may play key roles in the progression of HCV-associated hepatic diseases

Enhanced seed production under prolonged heat stress conditions in Arabidopsis thaliana plants deficient in cytosolic ascorbate peroxidase 2

Article discussing enhanced seed production under prolonged heat stress conditions in Arabidopsis thaliana plants deficient in cytosolic ascorbate peroxidase 2

Enhanced seed production under prolonged heat stress conditions in Arabidopsis thaliana

Reactive oxygen species play a key role in the response of plants to abiotic stress conditions. Their level is controlled in Arabidopsis thaliana by a large network of genes that includes the H(2)O(2)-scavenging enzymes cytosolic ascorbate peroxidase (APX) 1 and 2. Although the function of APX1 has been established under different growth conditions, genetic evidence for APX2 function, as well as for the mode of cooperation between APX1 and APX2, is very limited. This study characterized the response of Arabidopsis mutants deficient in APX1, APX2, and APX1/APX2 to heat, salinity, light, and oxidative stresses. The findings reveal that deficiency in APX2 resulted in a decreased tolerance to light stress, as well as an enhanced tolerance to salinity and oxidative stresses. Interestingly, plants lacking APX2 were more sensitive to heat stress at the seedling stage, but more tolerant to heat stress at the reproductive stage. Cooperation between APX1 and APX2 was evident during oxidative stress, but not during light, salinity, or heat stress. The findings demonstrate a role for APX2 in the response of plants to light, heat, salinity, and oxidative stresses. The finding that plants lacking APX2 produced more seeds under prolonged heat stress conditions suggests that redundant mechanisms activated in APX2-deficient plants during heat stress play a key role in the protection of reproductive tissues from heat-related damage. This finding is very important because heat-associated damage to reproductive tissues in different crops is a major cause for yield loss in agriculture production worldwide

The CC chemokine ligand (CCL) 1, upregulated by the viral transactivator Tax, can be downregulated by minocycline: possible implications for long-term treatment of HTLV-1-associated myelopathy/tropical spastic paraparesis

Abstract Background Chemokine (C-C motif) ligand 1 (CCL1) is produced by activated monocytes/ macrophages and T-lymphocytes, and acts as a potent attractant for Th2 cells and a subset of T-regulatory (Treg) cells. Previous reports have indicated that CCL1 is overexpressed in adult T-cell leukemia cells, mediating an autocrine anti-apoptotic loop. Because CCL1 is also known as a potent chemoattractant that plays a major role in inflammatory processes, we investigated the role of CCL1 in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Results The results showed that: (1) CCL1 was preferentially expressed in HAM/TSP-derived HTLV-1-infected T-cell lines, (2) CCL1 expression was induced along with Tax expression in the Tax-inducible T-cell line JPX9, (3) transient Tax expression in an HTLV-1-negative T-cell line activated the CCL1 gene promoter, (4) plasma levels of CCL1 were significantly higher in patients with HAM/TSP than in HTLV-1-seronegative patients with multiple sclerosis and HTLV-1-infected asymptomatic healthy carriers, and (5) minocycline inhibited the production of CCL1 in HTLV-1-infected T-cell lines. Conclusions The present results suggest that elevated CCL1 levels may be associated with the pathogenesis of HAM/TSP. Although further studies are required to determine the in vivo significance, minocycline may be considered as a potential candidate for the long-term treatment of HAM/TSP via its anti-inflammatory effects, which includes the inhibition of CCL1 expression