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.     

NPH3- and PGP-like genes are exclusively expressed in the apical tip region essential for blue-light perception and lateral auxin transport in maize coleoptiles

Phototropic curvature results from differential growth on two sides of the elongating shoot, which is explained by asymmetrical indole-3-acetic acid (IAA) distribution. Using 2 cm maize coleoptile segments, 1st positive phototropic curvature was confirmed here after 8 s irradiation with unilateral blue light (0.33 μmol m(-2) s(-1)). IAA was redistributed asymmetrically by approximately 20 min after photo-stimulation. This asymmetric distribution was initiated in the top 0-3 mm region and was then transmitted to lower regions. Application of the IAA transport inhibitor, 1-N-naphthylphthalamic acid (NPA), to the top 2 mm region completely inhibited phototropic curvature, even when auxin was simultaneously applied below the NPA-treated zone. Thus, lateral IAA movement occurred only within the top 0-3 mm region after photo-stimulation. Localized irradiation experiments indicated that the photo-stimulus was perceived in the apical 2 mm region. The results suggest that this region harbours key components responsible for photo-sensing and lateral IAA transport. In the present study, it was found that the NPH3- and PGP-like genes were exclusively expressed in the 0-2 mm region of the tip, whereas PHOT1 and ZmPIN1a, b, and c were expressed relatively evenly along the coleoptile, and ZmAUX1, ZMK1, and ZmSAURE2 were strongly expressed in the elongation zone. These results suggest that the NPH3-like and PGP-like gene products have a key role in photo-signal transduction and regulation of the direction of auxin transport after blue light perception by phot1 at the very tip region of maize coleoptiles.     

Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.     

TNF inhibited the apoptosis by activation of Akt serine/threonine kinase in the human head and neck squamous cell carcinoma

Tumour necrosis factor (TNF) is known to induce apoptosis, but recently, TNF was shown to promote cell survival, a process regulated by phosphatidylinositol-3-OH kinase (PI3K) and the NFkappaB pathway. In this study, we investigated the relationship between the molecules implicated in regulating TNF-induced cell survival and apoptosis induced by TNF in a human head and neck squamous cell carcinoma cell line (SAS), with special reference to the Akt pathway, one of the pathways related to cell survival. In SAS cells, TNF induced the phosphorylation of Akt at both Ser473 and Thr308, causing the activation of Akt, and also induced the phosphorylation and degradation of IkappaB (inhibitor of NFkappaB). This phosphorylation and degradation was inhibited by pretreating the cells with the PI3K inhibitors, wortmannin or LY294002. The apoptosis of SAS cells induced by TNF was dependent on the concentration: a high concentration of TNF, but not a low concentration, induced apoptosis within 30 h. However, a low concentration of TNF in the presence of wortmannin or LY294002 induced apoptosis. Furthermore, expression of the kinase-negative form of Akt, IKKalpha or IKKbeta, and the undegradable mutant of IkappaB, also induced apoptosis at low concentrations of TNF. When the SAS cells expressed constitutively activated Akt, apoptosis was not induced, even by high concentrations of TNF. These observations suggest that, in the SAS cell line, the PI3K-NFkappaB pathway contributes to TNF-induced cell survival and that inhibition of this pathway accelerates apoptosis.     

TRF1 Ensures the Centromeric Function of Aurora-B and Proper Chromosome Segregation

A cancer is a robustly evolving cell population originating from a normal diploid cell. Improper chromosome segregation causes aneuploidy, a driving force of cancer development and malignant progression. Telomeric repeat binding factor 1 (TRF1) has been established as a telomeric protein that negatively regulates telomere elongation by telomerase and promotes efficient DNA replication at telomeres. Intriguingly, overexpression of a mitotic kinase, Aurora-A, compromises efficient microtubule-kinetochore attachment in a TRF1-dependent manner. However, the precise role of TRF1 in mitosis remains elusive. Here we demonstrate that TRF1 is required for the centromeric function of Aurora-B, which ensures proper chromosome segregation. TRF1 depletion abolishes centromeric recruitment of Aurora-B and loosens sister centromere cohesion, resulting in the induction of merotelic kinetochore attachments, lagging chromosomes, and micronuclei. Accordingly, an absence of TRF1 in human and mouse diploid cells induces aneuploidy. These phenomena seem to be telomere independent, because a telomere-unbound TRF1 mutant can suppress the TRF1 knockdown phenotype. These observations indicate that TRF1 regulates the rigidity of the microtubule-kinetochore attachment, contributing to proper chromosome segregation and the maintenance of genomic integrity.     

Metabolomic analysis of the effects of omeprazole and famotidine on aspirin-induced gastric injury

Gastric mucosal ulceration and gastric hemorrhage are frequently associated with treatment by non-steroid anti-inflammatory drugs (NSAIDs); however, no convenient biomarker-based diagnostic methods for these adverse reactions are currently available, requiring the use of endoscopic evaluation. We recently reported five biomarker candidates in serum which predict gastric injury induced by NSAIDs in rats, but were unable to clarify the mechanism of change in the levels of these biomarker candidates. In this study, we performed capillary electrophoresis–mass spectrometry-based metabolomic profiling in stomach and serum from rats in which gastric ulcer was induced by aspirin and prevented by co-administration of omeprazole and famotidine. Results showed drug-induced decreases in the levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, and O-acetyl carnitine in all animals administered aspirin. In contrast, aspirin-induced decreases in the level of 4-hydroxyproline were suppressed by co-administration of omeprazole and famotidine. We consider that these changes were due to the prevention of gastric ulcer and decrease in the amount of collagen in stomach tissue by omeprazole and famotidine, without prevention of the NSAID-induced depression of mitochondrial function. In addition, the decreases in 4-hydroxyproline in the stomach was also detectable as changes in the serum. While further study is needed to clarify limitations of indications and extrapolation to humans, this new serum biomarker candidate of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.     

L1cam Is Crucial for Cell Locomotion and Terminal Translocation of the Soma in Radial Migration during Murine Corticogenesis

L1cam (L1) is a cell adhesion molecule associated with a spectrum of human neurological diseases, the most well-known being X-linked hydrocephalus. Although we recently demonstrated that L1 plays an important role in neuronal migration during cortical histogenesis, the mechanisms of delayed migration have still not been clarified. In this study, we found that cell locomotion in the intermediate zone and terminal translocation in the primitive cortical zone (PCZ) were affected by L1-knockdown (L1-KD). Time-lapse analyses revealed that L1-KD neurons produced by in utero electroporation of shRNA targeting L1 (L1-shRNAs) molecules showed decreased locomotion velocity in the intermediate zone, compared with control neurons. Furthermore, L1-KD neurons showed longer and more undulated leading processes during translocation through the primitive cortical zone. The curvature index, a quantitative index for curvilinearity, as well as the length of the leading process, were increased, whereas the somal movement was decreased in L1-KD neurons during terminal translocation in the PCZ. These results suggest that L1 has a role in radial migration of cortical neurons.     

Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)⁺CD45^– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl₄). Because the vast majority of EGFP⁺CD45^– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP⁺ PaSCs were also observed in CCl₄-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl₄-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C^high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP⁺F4/80⁺CCR2⁺ monocytic cells and EGFP⁺ PaSCs in the pancreas of CCl₄-treated chimeric mice receiving EGFP⁺ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP⁺ PaSCs in injured mice. We propose that CCR2⁺ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.