Aberrant histone acetylation contributes to elevated interleukin-6 production in rheumatoid arthritis synovial fibroblasts

Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA.     

Neurogenin2‐d4Venus and Gadd45g‐d4Venus transgenic mice: Visualizing mitotic and migratory behaviors of cells committed to the neuronal lineage in the developing mammalian brain

To achieve highly sensitive and comprehensive assessment of the morphology and dynamics of cells committed to the neuronal lineage in mammalian brain primordia, we generated two transgenic mouse lines expressing a destabilized (d4) Venus controlled by regulatory elements of the Neurogenin2 (Neurog2) or Gadd45g gene. In mid‐embryonic neocortical walls, expression of Neurog2‐d4Venus mostly overlapped with that of Neurog2 protein, with a slightly (1 h) delayed onset. Although Neurog2‐d4Venus and Gadd45g‐d4Venus mice exhibited very similar labeling patterns in the ventricular zone (VZ), in Gadd45g‐d4Venus mice cells could be visualized in more basal areas containing fully differentiated neurons, where Neurog2‐d4Venus fluorescence was absent. Time‐lapse monitoring revealed that most d4Venus⁺ cells in the VZ had processes extending to the apical surface; many of these cells eventually retracted their apical process and migrated basally to the subventricular zone, where neurons, as well as the intermediate neurogenic progenitors that undergo terminal neuron‐producing division, could be live‐monitored by d4Venus fluorescence. Some d4Venus⁺ VZ cells instead underwent nuclear migration to the apical surface, where they divided to generate two d4Venus⁺ daughter cells, suggesting that the symmetric terminal division that gives rise to neuron pairs at the apical surface can be reliably live‐monitored. Similar lineage‐committed cells were observed in other developing neural regions including retina, spinal cord, and cerebellum, as well as in regions of the peripheral nervous system such as dorsal root ganglia. These mouse lines will be useful for elucidating the cellular and molecular mechanisms underlying development of the mammalian nervous system.     

Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2-lysophospholipids in biological samples by LC-MS/MS

Lysophospholipids (LysoGPs) serve as lipid mediators and precursors for synthesis of diacyl phospholipids (GPs). LysoGPs detected in cells have various acyl chains attached at either the sn-1 or sn-2 position of the glycerol backbone. In general, acyl chains at the sn-2 position of 2-acyl-1-LysoGPs readily move to the sn-1 position, generating 1-acyl-2-lyso isomers by a nonenzymatic reaction called intra-molecular acyl migration, which has hampered the detection of 2-acyl-1-LysoGPs in biological samples. In this study, we developed a simple and versatile method to separate and quantify 2-acyl-1- and 1-acyl-2-LysoGPs. The main point of the method was to extract LysoGPs at pH 4 and 4°C, conditions that were found to completely eliminate the intra-molecular acyl migration. Under the present conditions, the relative amounts of 2-acyl-1-LysoGPs and 1-acyl-2-LysoGPs did not change at least for 1 week. Further, in LysoGPs extracted from cells and tissues under the present conditions, most of the saturated fatty acids (16:0 and 18:0) were found in the sn-1 position of LysoGPs, while most of the PUFAs (18:2, 20:4, 22:6) were found in the sn-2 position. Thus the method can be used to elucidate the in vivo role of 2-acyl-1-LysoGPs.     

Alternative Mammalian Target of Rapamycin (mTOR) Signal Activation in Sorafenib-resistant Hepatocellular Carcinoma Cells Revealed by Array-based Pathway Profiling

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.Hepatocellular carcinoma (HCC)¹ is the third most common cause of cancer-related death worldwide (1). Advanced HCC often cannot be managed with local treatments (surgical resection, ethanol injection, radiofrequency ablation, chemoembolization), but no systemic chemotherapy with conventional cytotoxic agents had been shown to be effective until a landmark phase III clinical trial (the Sorafenib HCC Assessment Randomized Protocol) revealed significant survival prolongation in patients treated with sorafenib (Nexavar; Bayer Healthcare Pharmaceuticals Inc. Berlin, Germany) (2). Furthermore, it has been reported that some patients show remarkable tumor shrinkage after short-term administration of sorafenib (3). Based on these results, sorafenib monotherapy has been employed as the current standard first-line treatment for unresectable HCC. However, not all HCC patients show the desired therapeutic benefits of sorafenib. The overall survival prolongation of unselected patients in the SHARP trial was limited to 2.8 months (2), and an objective tumor response was observed only in a small proportion of patients (0.6% to 2%) (2, 4). Given the relatively high cost and occasional severe adverse events (diarrhea, hand-foot skin reaction, hypertension, and others) (2, 4), there is an urgent need to identify a predictive biomarker that could exclude advanced HCC patients who are unlikely to benefit from sorafenib therapy.Sorafenib is a multi-kinase inhibitor that blocks tumor cell proliferation and angiogenesis through the inhibition of c-RAF and b-RAF, as well as many receptor tyrosine kinases, including vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor-α, Fms-related tyrosine kinase 3, RET, and c-KIT (5). In view of this broad inhibitory spectrum, the precise mechanisms underlying the anti-tumor activity remain elusive. To date, factors that have been identified as correlated with the efficacy of sorafenib include phosphorylated extracellular signal-regulated kinase 1 (p-ERK) (6), serum des-γ-carboxyprothrombin level (7), phosphorylated c-Jun protein (8), and fibroblast growth factor-3/4 gene amplification (3), but their clinical utility as predictive biomarkers has not been established.In the present study, we developed a new technique, high-density fluorescence reverse-phase protein array (RPPA), and used it to search for a biomarker that would identify patients in whom sorafenib would be effective, employing a large library of phosphorylation-site-specific antibodies. RPPA represents an emerging technology for proteomics, and it is well suited for the profiling of phosphorylated proteins. It involves micro-format dot immunoblotting of lysates from tissues or cells (9), allowing simultaneous monitoring of the expression of a particular phosphoprotein in hundreds to thousands of samples under identical conditions in a highly quantitative manner (10). In this study we profiled the activation status of 180 key signaling nodes across a panel of 23 HCC cell lines and identified de novo activation of mTOR signaling in sorafenib-resistant HCC cells.     

Regulation of a pentameric ligand-gated ion channel by a semiconserved cationic lipid-binding site

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure–function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABA_A receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABA_A receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs.     

Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis

Angiopoietin-like protein family 4 (Angptl 4) has been shown to regulate lipoprotein metabolism through the inhibition of lipoprotein lipase (LPL). We generated ApoE^−/−Angptl 4^−/− mice to study the effect of Angptl 4 deficiency on lipid metabolism and atherosclerosis. Fasting and postolive oil-loaded triglyceride (TG) levels were largely decreased in ApoE^−/−Angptl 4^−/− mice compared with and ApoE^−/−Angptl 4^+/+ mice. There was a significant (75 ± 12%) reduction in atherosclerotic lesion size in ApoE^−/−Angptl 4^−/− mice compared with ApoE^−/− Angptl 4^+/+ mice. Peritoneal macrophages, isolated from Angptl 4^−/− mice to investigate the foam cell formation, showed a significant decrease in newly synthesized cholesteryl ester (CE) accumulation induced by acetyl low-density lipoprotein (acLDL) compared with those from Angptl 4^+/+ mice. Thus, genetic knockout of Angptl 4 protects ApoE^−/− mice against development and progression of atherosclerosis and strongly suppresses the ability of the macrophages to become foam cells in vitro.     

Water and salt movement in soil driven by crop roots: a controlled column study

Water deficit and salt accumulation in soil presents serious problems to crop production in semi-arid regions. These problems depend on the active transpiration stream and the selective absorption of ions by crop roots. In this study, a large sized soil column system was used to examine the dynamics of water and ion transport and salt accumulation in soil layers. Special reference was placed on the effects of the active and selective absorption by roots of different crops (i.e., corn plants, sunflower plants and no plants). The column system was equipped with on-line systems for the control of groundwater level. Soil water content sensors enabled time-course evaluations of the volumetric water content and hence upward flux of the groundwater in the soils at different depths. Furthermore, the distribution and accumulation of ions in soil layers, plant organs and xylem sap were analyzed using ion chromatography. In this column experiment, diurnal and longer term changes in water movement and ion accumulation in soil, affected by root absorption characteristics of plants, were evaluated quantitatively. The results demonstrated that the column system was applicable for the quantitative analysis of the effects of root absorption by different crops on water deficit and salinization in soils.     

Establishment and characterization of hepatic stem-like cell lines from normal adult rat liver

The liver is a unique organ with the potential to regenerate from injury. Hepatic stem cells contribute to liver regeneration when surviving hepatocytes in injured liver are unable to proliferate. To investigate the mechanism of liver regeneration in vitro, we established hepatic stem cell lines named HY1, HY2 and HY3, derived from a healthy liver of adult rat. HY cells showed an expression pattern similar to oval cells, and efficiently induced hepatic differentiation following sequential treatment with type I collagen, transforming growth factor-beta1 (TGF-beta1), and hepatocyte growth factor (HGF) or oncostatin M (OSM). These results suggested that HY cells are liver stem cells representing an excellent tool for in vitro studies on liver regeneration.     

Depletion of Selenoprotein GPx4 in Spermatocytes Causes Male Infertility in Mice

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674–683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a Cre-loxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.     

Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713

Cryphonectria hypovirus 1 (CHV1), associated with the picorna-like superfamily, infects the chestnut blight fungus and attenuates the virulence of the host fungus. The genomic RNA of the virus has two continuous open reading frames, A and B, separated by the pentanucleotide UAAUG. We present here evidence suggesting that ORF B is translated from genome-sized virus mRNA by a coupled termination/reinitiation mechanism mediated by the pentamer. In the coupled translation, the overlapping UAA and AUG triplets serve as the stop codon of ORF A and the initiator of ORF B, respectively. This was established by the use of a luciferase assay with a basic construct containing the ORF A sequence and the firefly luciferase gene while retaining the pentamer between the two coding sequences. The proportion of ribosomes reinitiating translation after terminating was determined to be 2.5–4.4% by three independent assay systems in fungal and insect cells. Use of a series of mutant constructs identified two sequence elements, the pentamer and the p40 sequence, that affect the efficiency of coupled translation and virus replication. Together, these results provide the first example of coupled translation facilitated by the pentanucleotide UAAUG in the kingdom Fungi. The mechanism by which the preceding p40-coding sequence promotes reinitiation is discussed.