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Climatic changes due to emission of greenhouse gases are a global concern. These emissions occur by combustion of fossil fuels whose drought is near in which case renewable energy is the only alternative. Microalgae are promising sources of sustainable bioenergy production, and utilisation of wastewater as cultures is recommended for economical production cost. In this study, indigenous microalgae, which had adaptability for wastewater samples, were cultivated with a municipal secondary effluent, and influences of changes in inorganic nitrogen (IN) concentration, specifically IN increase, on temporal accumulation and degradation of organic components in indigenous microalgae were investigated. Indigenous microalgae accumulated total lipids and carbohydrates against reduced IN, and increase in superoxide dismutase suggested that the accumulation was possibly induced by generating reactive oxygen species. Continued cultivation of indigenous microalgae under the IN exhausted condition should be avoided because of the resulting total carbohydrate degradation. IN replenishment when IN was decreased but still existed in the culture and that when IN was exhausted in the culture triggered sharp degradation of the total carbohydrate, which possibly utilised to accumulate crude protein and/or chlorophyll a for continuous growth or regrowth. The total carbohydrate was accumulated and recovered after the degradation; meanwhile, two or three days were required for the recovery of the total carbohydrate. In addition, the IN replenishment also resulted in total lipid degradation. Therefore, to produce indigenous microalgae with high and stable total carbohydrate and lipid content, it was critical to prevent IN increase in the culture.

Cultivation conditions influence microalgal cellular components, such as lipid accumulation under nutrient depletion, high light irradiation and salinity stress. In this study, indigenous microalgal consortia were cultivated in batch mode using an actual treated effluent. The temporal response of cellular components to the variations in nitrogen concentration and influence of light irradiation on the response were investigated. Prolonged exposure of indigenous microalgal consortia to nitrogen exhaustion had minor effects on total lipid accumulation and enhancement of energy content. Nitrogen replenishment was followed by immediate crude protein accumulation for growth recovery. Total lipid reduction was observed under light and dark conditions after nitrogen replenishment. A one-day lag after nitrogen replenishment in the total lipid reduction was revealed under nitrogen depletion; meanwhile, under nitrogen exhaustion, lipids were utilised as the primary carbon and/or energy source after replenishment, as represented by the decrease from 10.8% to 9.04% within 6 h after the replenishment.

Aims We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. Methods and results The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. Conclusion Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

We previously reported that impaired retinoid signaling causes hepatocellular carcinoma (HCC) through oxidative stress. However, the interaction between oxidative stress and retinoid signaling has not been fully understood. To address this issue, the effects of hydrogen peroxide on the transcriptional activity of RAR/RXR heterodimers, RARα and RXRα proteins and intracellular signaling pathways were examined. The transcriptional activity of RAR/RXR examined by the DR5-tk-Luc reporter assay was significantly suppressed. The RARα protein level began to decrease at 6 h after treatment and declined thereafter. However, RARα mRNA were not changed. Activation of extracellular regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and Akt was observed after treatment of hydrogen peroxide. SP600125, an inhibitor of JNK, reversed the RARα protein level reduced by hydrogen peroxide. Anisomycin, an activator of JNK, reduced RARα protein. Transfection of wild-type JNK-constitutive actively expressing plasmid, but not kinase-negative JNK-expressing plasmid caused reduction of RARα protein. Proteasomal degradation of RARα was observed after anisomycin treatment; however, the mutant RARα, of which phosphorylation sites are replaced with alanines, was not degradated. In hepatitis C virus (HCV)-related human liver tissues, phospho-JNK and RARα reciprocally expressed with the progression of liver disease. Finally, the staining of 8-OHdG and thioredoxin was increased with the disease progression. These data indicate that JNK activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of RARα, suggesting that a vicious cycle between aberrant retinoid signaling and oxidative stress accelerates hepatocarcinogenesis.

Aim: We have recently reported that hyperdynamic state of retinoid metabolism, which may lead to the shortage of retinoid, is observed in patients with non-alcoholic fatty liver disease (NAFLD). Hepatic iron overload, which causes production of reactive oxygen species (ROS), is also frequently seen in NAFLD patients. The aim of the study is to examine iron state and retinoid metabolic state simultaneously, and to clarify the relationship between two disorders. Methods: Thirty-six persons, comprising 17 patients with simple steatosis (SS), 11 with NASH, and 8 normal controls (N), were examined on hepatic expression of iron metabolism-related genes including hemojuvelin (HJV), hepcidin (HEPC), transferrin receptor 1 and 2 (TfR1, TfR2), ferroportin (FPN), neogenin (NEO) and ferritin heavy chain (FtH) and hepatic iron contents in addition to expression 51 genes which is involved in retinoid metabolism and antioxidative action. Results: In patients with NAFLD, expression of HJV, TfR2, FPN, TfR1, FtH, SOD and catalase was increased, compared with that in N. In addition, hepatic iron content, which was increased in NASH, was correlated with expression level of TfR2. Expression of cellular retinoid binding protein (CRBP1), alcohol dehydrogenase 1 (ADH1) and cytochrome P450 26A1(CYP26A1) was significantly correlated with that of HJV, TfR2 and FPN, respectively. Conclusion: The results of the present study suggest that the reasons responsible for iron accumulation in NASH in the present study may partly be due to enhanced expression of TfRs, especially TfR2, and hyperdynamic state of retinoid metabolism is closely related to iron metabolism in the disease.

Aim: The patients with non-alcoholic fatty liver disease (NAFLD) have been reported to be at greater risk for progression to chronic liver disease including liver cirrhosis (LC). To examine the mechanisms for the progression of NAFLD, a genetic analysis of hepatic expression profile in retinoid metabolism in NAFLD was performed since the loss of retinoid signaling is associated with the progression of liver disease via reactive oxygen species (ROS) generation. Methods: Fifty-one genes, which are associated with retinoid metabolism and action, were examined in thirty six subjects including 17 patients with simple steatosis, 11 with non-alcoholic steatohepatitis (NASH) and eight controls were examined by real-time reverse transcriptase polymerase chain reaction. Immunohistochemical study was also done by 3 kinds of antibodies. Results: Higher expression of CRBP1 LRAT, DGT1/2 and CES1 in NAFLD suggests that mutual conversion between retinyl ester and retinal occurs actively. Expression of ADH1/2/3, RDH5/10/11, DHRS3 and RALDH1/3 was increased in NAFLD, suggesting that oxidation process from retinol to all-trans retinoic acid (ATRA) was enhanced. Importantly, greater expression of CYP26A1 indicated that degradation of ATRA was enhanced in NAFLD. Further, expression of SOD1/2, catalase, thioredoxin and uncoupling protein 2 was also enhanced. Conclusion: Hyperdynamic state of retinoid metabolism is present in the liver tissues with NAFLD, which may be a putative mechanism by which NAFLD progresses to chronic liver disease including LC.

Hepatic iron overload has been frequently observed in the liver of patients with chronic liver diseases. In this study, the effect of hepatic fatty acid accumulation on the iron metabolism was investigated. Mice fed a choline-deficient diet developed severe steatosis associated with increased total amount of non-heme iron in the liver. Hepatic lipid contents were well correlated with the iron amount. The choline-deficient diet significantly downregulated hepcidin while increases in hemojuvelin and transferrin receptor 2 and a decrease in Tmprss6 expression were observed. Moreover, ferroportin expression was downregulated in the livers of choline-deficient mice while increases in transferrin receptor 1 and divalent metal transporter 1 and a decrease in ferritin expression were observed in accordance with increased hepatic iron content. The expression of hepcidin and ferroportin mRNA was negatively correlated to hepatic lipid concentrations. These results suggest that enhanced dietary iron intake and reduced hepatic iron efflux occur in the mice fed a choline-deficient diet. In addition, a possible link between hepatic iron and lipid metabolism is also suggested.

We investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the stability of Kv1.5 channel protein. The expression and function of Kv1.5 (Kv1.5-FLAG) in transfected African green monkey kidney fibroblast cells as well as rat atrium were estimated by immunoblotting, immunoprecipitation, immunofluorescence and patch-clamp techniques. Both EPA and DHA immediately blocked Kv1.5 channel current in a dose-dependent manner, accompanied by reduction of their phosphorylation. Chronic treatment (for 12 h) with EPA at lower concentrations (0.3-10 muM) increased the level of Kv1.5-FLAG protein as well as Kv1.5 channel current without changes in its gating kinetics, prolonging its half-life; in contrast, both EPA and DHA at higher concentrations (30-100 muM) decreased the expression of Kv1.5-FLAG. EPA at the higher concentrations also decreased mRNA of Kv1.5 and synapse-associated protein 97 expression. EPA at the lower concentrations increased Kv1.5 expression in the endoplasmic reticulum, Golgi apparatus and cell membrane. EPA-induced increase of Kv1.5 channel expression and current was abolished by pretreatment with the protein transport inhibitor brefeldin A or colchicines, and by the Kv1.5 channel blocker 4-aminopyridine. Oral administration of EPA (30 mg/kg) increased the level of endogenous Kv1.5 in rat atria. These results indicate that chronic treatment with EPA at lower concentrations stabilizes Kv1.5 channel protein in the endoplasmic reticulum and Golgi apparatus thereby enhancing the Kv1.5 channel current on the cell membrane.

The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.

Human bone marrow–derived mesenchymal stem cells (BM-MSCs) are expected to be a potential source of cells for transplantation. Although recent reports have shown that isolated MSCs can differentiate into hepatocytes, the efficiency of differentiation is insufficient for therapeutic application. To circumvent this problem, it is necessary to understand the mechanisms of hepatic differentiation of human BM-MSCs. Hepatocyte nuclear factor 3β (HNF3β), a forkhead/winged helix transcription factor, is essential for liver development. In the present study, we established a tetracycline (Tet)-regulated expression system for HNF3β in UE7T-13 BM-MSCs. HNF3β expression significantly enhanced expression of albumin, α-fetoprotein (AFP), tyrosine amino transferase (TAT) and epithelial cell adhesion molecule (EpCAM) genes. The differentiated cells showed hepatocyte-specific functions including glycogen production and urea secretion. During treatment with the Tet-on system for 8 days, over 80% of UE7T-13 cells turned out to express albumin. Furthermore, the combination of Tet with basic fibroblast growth factor (bFGF) efficiently induced the genes such as albumin and TAT, which are associated with maturity of hepatocytes; however, it suppressed genes such as AFP and EpCAM, which are associated with immaturity of hepatocytes, suggesting that Tet-induced HNF3β expression sensitizes BM-MSCs to bFGF signals. Finally, the results of the present study suggest that down-regulation of Wnt/β-catenin signals caused by translocation of β-catenin to cytoplasmic membrane is associated with hepatic differentiation of human BM-MSCs. Conclusion: HNF3β expression induced efficient differentiation of UE7T-13 human BM-MSCs. (HEPATOLOGY 2008;48:597–606.)

Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to be an excellent source of cells for transplantation. In addition, the stem cell plasticity of human UCBMSCs, which can transdifferentiate into hepatocytes, has been reported. However, the mechanisms involved remain to be clarified. To identify the genes and/or signals that are important in specifying the hepatic fate of human UCBMSCs, we analyzed gene expression profiles during the hepatic differentiation of UCBMSCs with human telomerase reverse transcriptase, UCBMSCs immortalized by infection with a retrovirus carrying telomerase reverse transcriptase, but whose differentiation potential remains unchanged. Efficient differentiation was induced by 5-azacytidine (5-aza)/hepatocyte growth factor (HGF)/oncostatin M (OSM)/fibroblast growth factor 2 (FGF2) treatment in terms of function as well as protein expression: 2.5-fold increase in albumin, 4-fold increase in CCAAT enhancer-binding protein alpha, 1.5-fold increase in cytochrome p450 1A1/2, and 8-fold increase in periodic acid-Schiff staining. Consequently, we found that the expression of Wnt/beta-catenin-related genes downregulated, and the translocation of beta-catenin was observed along the cell membrane and in the cytoplasm, although some beta-catenin was still in the nucleus. Downregulation of Wnt/beta-catenin signals in the cells by Fz8-small interference RNA treatment, which was analyzed with a Tcf4 promoter-luciferase assay, resulted in similar hepatic differentiation to that observed with 5-azacytidine/HGF/OSM/FGF2. In addition, the subcellular distribution of beta-catenin was similar to that of cells treated with 5-azacytidine/HGF/OSM/FGF2. In conclusion, the suppression of Wnt/beta-catenin signaling induced the hepatic differentiation of UCBMSCs, suggesting that Wnt/beta-catenin signals play an important role in the hepatic fate specification of human UCBMSCs.

Since rebamipide is effective for the treatment of ulcerative colitis (UC), we examined the involvement of hepatocyte growth factor (HGF) in the action of rebamipide.Fifty-five and forty female Balb/c mice, respectively, were used in Exp. 1 and 2.50 mg/kg/day rebamipide (Exp. 1) and 1 x 10(7) pfu pAxCAHGF (the CAG promoter-driving HGF gene in adenovirus vector) (Exp. 2) were intrarectally introduced after induction of colitis by 4 % dextran sulfate sodium (DSS).Therapeutic effects were assessed by cell proliferation and apoptosis.Rebamipide caused proliferation of epithelial cells at 10 days after treatment, and decreased apoptosis at 10, 14 and 21 days, compared with controls. Expression of HGF was greatly increased in rebamipide-treated mice. pAxCAHGF caused cell proliferation and apoptosis, which showed the same pattern as with rebamipide treatment.Rectal administration of rebamipide is effective for DSS-induced colitis in association with induction of HGF.

As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.

Background Remote reperfusion lung injury occurs in patients with vascular occlusion and surgical procedures. Inosine monophosphate (IMP) produced by adenosine monophosphate deaminase (AMPD) 3 is involved in the remote reperfusion injury. The purpose of the present study was to identify whether IMP administration attenuated the remote reperfusion lung injury in a skeletal muscle ischemia-reperfusion model. Methods and Results A remote reperfusion lung injury was created using reperfusion after the bilateral ligation of the hind-limb. AMPD activity, myeloperoxidase (MPO) activity, IMP, AMPD3 mRNA and tumor necrosis factor (TNF)-α in the lungs before and after reperfusion were analyzed. Furthermore, the effects of IMP on these parameters were examined. AMPD3 mRNA, AMPD activity and IMP production in the lungs significantly increased after ischemia-reperfusion with increases in MPO activity, TNF-α level and decreased oxygen saturation (SpO2). Histological examination of the lungs demonstrated significant neutrophil infiltration and accumulation. IMP administration significantly reduced MPO activity, TNF-α and neutrophil infiltration, with ameliorated SpO2. Conclusions Along with the activation of AMPD3, ischemia-reperfusion-induced lung inflammation is associated with increased MPO activity and TNF-α level. IMP significantly decreased the lung injury, MPO activity, TNF-α and increased SpO2. These findings may lead to the development of a new therapeutic strategy for remote reperfusion lung injury. (Circ J 2007; 71: 591 - 596)

Background The voltage-gated potassium channel Kv1.5 plays a critical role in the maintenance of the membrane potential. While protein degradation is one of the major mechanisms for the regulation of channel functions, little is known on the degradation mechanism of Kv1.5. Methods and results Kv1.5 was expressed in COS cells and its degradation, intracellular localization, and channel activities were assessed by pulse-chase analysis, immunofluorescence, and patch clamp techniques, respectively. Expressed Kv1.5 had a half-life time of ∼6.7 h, which was prolonged by the proteasome inhibitors of MG132, ALLN, proteasomal inhibitor 1, or lactacystine, but not by a lysosomal inhibitor chloroquine. MG132 increased the protein level of Kv1.5, as well as the level of its ubiquitinated form in a dose-dependent manner. Similar effects of MG132 on endogenous Kv1.5 were seen in cultured rat atrial cells. Within a cell, Kv1.5 was mainly localized in both the endoplasmic reticulum and Golgi apparatus. MG132 increased the immunoreactivity of Kv1.5 in these compartments and also increased Ikur currents through the cell-surface Kv1.5. Pretreatment with either brefeldin A or colchicine abolished MG132-induced increase in Ikur currents. Conclusion Kv1.5 is degraded by the proteasome. The inhibition of the proteasome increased Ikur currents secondary to stabilization of the channel protein in the endoplasmic reticulum/Golgi apparatus.

ATP-sensitive K+ channels (KATP:SUR2A+Kir6.2) play a pivotal role in cardiac protection against ischemia and reperfusion injury. When expressed in COS cells, Kir6.2 was short-lived with a half-life time of 1.9 h. The half-life time of Kir6.2 was prolonged by proteasome inhibitors MG132, ALLN, proteasome inhibitor 1, and lactacystine, but not at all by a lysosomal inhibitor chloroquine. MG132 also increased the level of ubiquitinated Kir6.2 without affecting its localization in the endoplasmic reticulum and Golgi apparatus. In electrophysiological recordings, MG132 augmented nicorandil-activated KATP currents in COS cells expressing SUR2A and Kir6.2 as well as the same currents in neonatal rat cardiomyocytes. Like MG132, a Na+ channel blocker aprindine prolonged the half-life time of Kir6.2 and augmented KATP. Finally, both aprindine and MG132 inhibited the 20S proteasome activity in vitro. These results suggest a novel activity of aprindine to enhance KATP currents by inhibiting proteasomal degradation of Kir 6.2 channels, which may be beneficial in the setting of cardiac ischemia.

Angiotensin II (Ang II) has been reported to indirectly influence atrial electrical activity and to play a critical role in atrial arrhythmias in hypertensive patients. However, it is unclear whether Ang II has direct effects on the electrophysiological activity of the atrium affected by hypertension. We examined the effects of Ang II on the action potentials of atrial myocytes enzymatically isolated from spontaneous hypertensive rats (SHRs). The action potentials were recorded by the perforated patch-clamp technique and the atrial expression of the receptors AT1a and AT2 was measured by radioimmunoassay. Ang II significantly shortened the action potential durations (APDs) of SHRs without changes in the resting membrane potentials (RMPs). Pretreatment with selective AT1a blockers abolished the Ang II-induced reduction of atrial APDs of SHRs; however, a selective AT2 blocker did not, which was consistent with the results of the receptor assay. Pretreatment with phosphatidylinositol 3 (PI3)-kinase inhibitor, phospholipase C inhibitor, or protein kinase C (PKC) inhibitor abolished the Ang II-induced shortening of atrial APDs, but pertussis toxin and protein kinase A (PKA) inhibitor did not. To study the effects of chronic AT1a inhibition on Ang II-induced shortening of atrial APD, SHRs were treated with AT1a blocker for 4 weeks. AT1a blocker abolished the Ang II-induced reduction of atrial APDs of SHRs and also significantly lowered their blood pressure. In conclusion, Ang II shortened atrial APDs of SHRs via AT1a coupled with the Gq-mediated inositol triphosphate (IP3)-PKC pathway. Our findings indicated that Ang II caused atrial arrhythmias in hypertensive patients by shortening the effective refractory period of the atrium.

Na+チャネル遮断薬ピルジカイニド (Pil) がKv1.5チャネル蛋白を含む半減期の短い蛋白の増加作用を有することを見出したので, その機序をリドカイン (Lido) と比較して報告する.PilとLidoはプロテアソームで分解されるp53やIKK2蛋白を用量依存性に安定化し, このp53蛋白への作用は変異Mdmによりユビキチン・プロテアソーム系が遮断されると生じない, PilとLidoは用量依存性に20Sプロテアソーム活性を抑制するが, l-verapamilやE-4031にはこの作用は認められなかった.さらにPilはLidoに比較してより低濃度 (臨床使用濃度) でp53蛋白を安定化した.同様にPilとLidoはプロテアソームで分解されるKv1.5チャネル蛋白を安定化して細胞膜での/Kurを増加し, この作用は発現された培養COS7細胞Kv1.5チャネルのみならずラツト心房筋Kv1.5チャネルでも認められた, またKv1.5チャネル蛋白の安定化作用はPilがLidoより強い.その分子機序をin silicoドッキング試験から検討し, PilとLidoはともに20sプロテアソームを構成するβ5サブユニットの基質結合部位に作用して, その活性を抑制すると推測でき, PilがLidoに比較して結合性が高いことも再現できた, これらの結果からNa+チヤネル遮断薬Pilが臨床使用濃度でプロテアソーム活性を抑制し, Kv1.5チャネル蛋白を含む半減期の短い蛋白を増加させる作用を発揮することが明らかにされた.

To study the tumorigenesis of Epstein–Barr virus (EBV)-positive epithelial cell lines GT38 and GT39 derived from human gastric tissues, we inoculated these cells under the skin of severe combined immunodeficient (SCID) mice. The development of tumors was observed in each of the mice about 2 months after the inoculation. The tumors were diagnosed with undifferentiated carcinoma by hematoxylin/eosin staining. EBV-encoded small RNA1 was detected in the paraffin-embedded tumor sections. The tumor cells had human chromosome. The circular, but not linear, EBV DNA was detected in the tumors. The molecular sizes of EBV DNA termini were the same as that of the inoculated GT38 or GT39 cells. The expressions of EBV nuclear antigen 2 and latent membrane protein 1 reduced in the tumors. Transcripts of Bam HI C and W promoters in latency III were detected in the tumors and the cultured cells in vitro. The tumor cells were passaged from one SCID mouse to other SCID mice and to cultures in vitro. This is the first evidence that the EBV-positive epithelial cell lines produced tumors in the SCID mouse.

We characterized the cell growth and Epstein-Barr virus (EBV) reactivation for EBV infected epithelial cell lines, GT38, GT39, and GTC-4 using 12-O-tetradecanoylphorbol-13-acetate (TPA). These cell lines grew similarly in liquid medium, and formed colonies in soft agar. The cell growth was inhibited with TPA, dose-dependently in liquid medium. The colony formation was enhanced with low concentrations of TPA, but was inhibited with high concentrations. The latent EBV was reactivated with high concentrations of TPA as shown by the expression of EBV BZLF1 gene product ZEBRA. The effects of TPA on GTC-4 were compared with a Burkitt's lymphoma cell line Raji. The mode of actions of TPA in GTC-4 was different from Raji in terms of cell growth and EBV reactivation. The effective concentrations of TPA for cell growth inhibition and EBV reactivation were higher in Raji than GTC-4. Cell cycle analysis showed that TPA (20 ng/ml) induced cell cycle arrest to Raji but not to GTC-4; however, the rate of trypan blue stained cells increased in the TPA treated GTC-4 but not Raji. These results demonstrated that TPA affects differentially for the stimulation and inhibition of cell growth, and also EBV reactivation depends on TPA concentrations and cell types.

To understand the mechanism by which Epstein-Barr virus (EBV) is activated in Akata cells by cross-linking of surface immunoglobulin, the interaction between mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and EBV activation was investigated. Immunoblotting using an anti-phosphoMAPK antibody (Ab) revealed that anti-IgG Ab induced rapid phosphorylation of MAPK in the cells. The phosphorylation was inhibited by MAPK/ERK kinase specific inhibitor, PD98059. The expressions of the EBV immediate early BZLF1 mRNA and its protein product ZEBRA, and early antigen were also inhibited by the inhibitor. These results indicate that MAPK is involved in the pathways of EBV activation.

We characterized the expression of Epstein–Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) orn-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cell lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with aXhoI probe in both cell lines. These epithelial cell lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

Remote reperfusion lung injury following skeletal muscle ischemia and reperfusion accounts for high morbidity and mortality. AMP deaminase (AMPD), a key enzyme for nucleotide cycle, has been implicated in the regulation of this phenomenon. However, the function of Ampd2 and Ampd3 subtype has not been elucidated in remote reperfusion rodent lung injury. We utilized AMPD3 and AMPD2-deficient mice. The two types of AMPD-deficient mice and wild-type (WT) littermates were subjected to ischemia-reperfusion injury. After 3h bilateral hind-limb ischemia and reperfusion, AMPD3 mRNA, AMPD activity and inosine monophosphate (IMP) increased significantly in WT and AMPD2-deficient mice lungs, while they did not show significant alterations in AMPD3-deficient mice lungs. Genetic inactivation of Ampd3 resulted in markedly accelerated myeloperoxidase (MPO) activity along with exaggerated neutrophils infiltration and hemorrhage in the lungs compared to WT and AMPD2-deficient mice, furthermore, IMP treatment significantly attenuated MPO activity and neutrophils infiltration in WT and the two types of AMPD-deficient mice lungs after 3h reperfusion. These findings demonstrate for the first time in AMP-deficient mice models that AMPD3 plays a critical role in remote reperfusion lung injury via generation of IMP and validate the potential to use IMP into the clinical arena to attenuate remote ischemia-reperfusion lung injury.

We have recently reported that hyperdynamic state of retinoid metabolism, which may lead to the shortage of retinoid, is observed in patients with non-alcoholic fatty liver disease (NAFLD). Hepatic iron overload, which causes production of reactive oxygen species (ROS), is also frequently seen in NAFLD patients. The aim of the study is to examine iron state and retinoid metabolic state simultaneously, and to clarify the relationship between two disorders. Thirty-six persons, comprising 17 patients with simple steatosis (SS), 11 with NASH, and 8 normal controls (N), were examined on hepatic expression of iron metabolism-related genes including hemojuvelin (HJV), hepcidin (HEPC), transferrin receptor 1 and 2 (TfR1, TfR2), ferroportin (FPN), neogenin (NEO) and ferritin heavy chain (FtH) and hepatic iron contents in addition to expression 51 genes which is involved in retinoid metabolism and antioxidative action. In patients with NAFLD, expression of HJV, TfR2, FPN, TfR1, FtH, SOD and catalase was increased, compared with that in N. In addition, hepatic iron content, which was increased in NASH, was correlated with expression level of TfR2. Expression of cellular retinoid binding protein (CRBP1), alcohol dehydrogenase 1 (ADH1) and cytochrome P450 26A1(CYP26A1) was significantly correlated with that of HJV, TfR2 and FPN, respectively. The results of the present study suggest that the reasons responsible for iron accumulation in NASH in the present study may partly be due to enhanced expression of TfRs, especially TfR2, and hyperdynamic state of retinoid metabolism is closely related to iron metabolism in the disease.

To clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs). Using human LX-2 HSC cells, we examined the effects of PPC on expression of α-smooth muscle actin (α-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor β1 (TGFβ1). PPC suppressed ROS which are induced by TGFβ1, phosphorylation of p38MAPK, and expression levels of α-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels. These results suggest that ROS and Nox4 induced by TGFβ1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.

The patients with non-alcoholic fatty liver disease (NAFLD) have been reported to be at greater risk for progression to chronic liver disease including liver cirrhosis (LC). To examine the mechanisms for the progression of NAFLD, a genetic analysis of hepatic expression profile in retinoid metabolism in NAFLD was performed since the loss of retinoid signaling is associated with the progression of liver disease via reactive oxygen species (ROS) generation.Fifty-one genes, which are associated with retinoid metabolism and action, were examined in thirty six subjects including 17 patients with simple steatosis, 11 with non-alcoholic steatohepatitis (NASH) and eight controls were examined by real-time reverse transcriptase polymerase chain reaction. Immunohistochemical study was also done by 3 kinds of antibodies.Higher expression of CRBP1 LRAT, DGT1/2 and CES1 in NAFLD suggests that mutual conversion between retinyl ester and retinal occurs actively. Expression of ADH1/2/3, RDH5/10/11, DHRS3 and RALDH1/3 was increased in NAFLD, suggesting that oxidation process from retinol to all-trans retinoic acid (ATRA) was enhanced. Importantly, greater expression of CYP26A1 indicated that degradation of ATRA was enhanced in NAFLD. Further, expression of SOD1/2, catalase, thioredoxin and uncoupling protein 2 was also enhanced.Hyperdynamic state of retinoid metabolism is present in the liver tissues with NAFLD, which may be a putative mechanism by which NAFLD progresses to chronic liver disease including LC.

Changes in the expression rate of epidermal growth factor receptor (EGFR) mRNA with age in the liver of rats were examined to assess the contribution of EGF to the proliferation of hepatocytes in younger animals. Northern blot analyses with a 32P-labeled probe derived from the extracellular domain of EGFR cDNA were evaluated quantitatively by normalizing the densitometric scanning data with the relative amounts of poly(A)+ RNA in the samples. The expression of EGFR mRNA was low during the early period and increased towards the adult level after 21 days of birth. In contrast, the rate of beta-actin mRNA expression was significantly higher immediately after birth and showed a tendency to decrease with time.

To identify gene products involved in castration-induced involution of the rat ventral prostate, we constructed a subtraction cDNA library of the ventral prostate from rats castrated for 48 h. The library was screened with subtracted cDNA probes enriched for sequences with a low copy number expressed in intact or castrated rats. As a result of differential screening, 48 cDNA clones representing 10 different induced mRNAs were isolated. The time course of these mRNA inductions after castration was examined. Within the first 24 h after castration, the level of mRNAs for these cDNA clones was significantly increased and it reached its peak by 48-72 h after castration. Although mRNAs for these cDNA clones were expressed in various tissues from intact rats, an increase in mRNA as a response to castration was observed only in the ventral prostate. Partial sequence analyses of the 10 cDNA clones indicate that three cDNA clones represent rat glutathione S-transferase Yb-1, Yb-2 and Yb-3 subunit mRNA sequences, but for others respective homologues could not be found in a search of the GenBank database (release 67).

A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1α) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.

Although the importance of reactive oxygen species (ROS) in the pathogenesis of various diseases is stressed, clinical significance of the markers reflecting DNA oxidation such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) remains to be clarified.To examine clinical usefulness of 8-OHdG in healthy individuals in comparison with liver disease patients, urinary excretion of 8-OHdG was measured in 336 healthy individuals and 110 patients with liver disease.In healthy persons, the 8-OHdG excretion was increased in an age-dependent manner. It was positively correlated with cigarettes smoked a day and negatively correlated with body mass index (BMI) (P0.05, each). Age, smoking and BMI were independent predictors of urinary 8-OHdG excretion (P0.01, P0.01 and P0.05, respectively). In liver disease, the excretion of 8-OHdG was not changed, as compared with healthy individuals. However, the liver disease patients under the age of 40 had higher values of 8-OHdG than healthy persons. In addition, the urinary excretion of 8-OHdG was higher in patients with hepatitis C virus (HCV) infection than those with hepatitis B virus (HBV) infection.The results of the present study suggest that measurement of urinary 8-OHdG excretion is useful in assessing DNA oxidation caused by aging, smoking, body composition and liver disease.

AIM Bone marrow-derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes. METHODS The culture conditions to induce the efficient differentiation of human bone marrow-derived UE7T-13 cells were examined using cytokines, hormones, 5-azacytidine and type IV collagen. RESULTS We found that combination of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) with type IV collagen coating induced hepatic differentiation of UE7T-13 cells at over 30% frequency, where expression of albumin mRNA was increased over 20-fold. The differentiated cells had functions of albumin production, glycogen synthesis and urea secretion as well as expressing hepatocyte-specific genes. In addition, these cellshave binuclear and cuboidal morphology, which is a characteristic feature of hepatocytes. During hepatic differentiation, UE7T-13 cells showed depressed expression of WISP1 and WISP2 genes, members of the CCN family. Conversely, knockdown of WISP1 or WISP2 gene by siRNA stimulated hepatic differentiation. The effect of aFGF/bFGF/HGF/type IV collagen coating and WISP1-siRNA on hepatic differentiation was additive. CONCLUSION The present study suggests that aFGF/bFGF/HGF/type IV collagen coating is the efficient condition for hepatic differentiation of UE7T-13 cells, and that WISP1 and WISP2 play an important role in hepatic transdifferentiation of these cells.

To rescue patients with severe liver injury, it is critical to develop the efficient regulatory system of hepatic stem cell proliferation in vitro. Our aims are to examine whether combination of adenovirus-mediated hepatocyte growth factor (HGF) gene transfer with signal transduction inhibitors can regulate cell proliferation of oval cells.We examined the effects of treatment with adenoviral mediated HGF gene transfer and signal transduction inhibitors including LY294002, rapamycin and U0126 on proliferation OC/CDE22 hepatic oval cells and expression of signal transduction molecules.Infection with pAxCAHGF expanded the cells by 8-fold at 2 days, by 18-fold at 3 days and by 55-fold at 4 days. The addition of inhibitors inhibited pAxCAHGF-induced cell proliferation by LY294002 or rapamycin (P0.01, each). U0126 also inhibited growth of hepatic oval cells (P0.01). pAxCAHGF treatment induced phosphorylation of AKT. Treatment with rapamycin resulted in enhanced phosphorylation of AKT, and phosphorylation of AKT was induced by pAxCAHGF plus U0126.Autocrine expression of HGF with signal transduction inhibitors can regulate proliferation of OC/CDE22 hepatic oval cells. In addition, the AKT pathway is important for HGF-stimulated hepatic oval cell proliferation.

A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame 1 (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vIL-10) gene was analyzed using PCR-direct sequencing in African Burkitt’s lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had China1-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, Bam HI or Eco RI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.

The mechanisms of prevention of the development of liver cancer by NIK-333, an acyclic retinoid (ACR), were investigated. The transgenic mice expressing the dominant negative form of retinoic acid receptor alpha (RARE mice), that produce reactive oxygen species and lead to development of liver tumor were used. The effect of NIK-333 on hepatocarcinogenesis in RARE mice was studied. The RARE mice were examined after feeding 0.03% and 0.06% NIK-333 diets at 12 months of age. In the mice fed 0.06% NIK-333 diet, tumor incidence was greatly suppressed, compared to that of wild type mice (0/9 versus 5/9, P0.05), but not in the mice fed 0.03% NIK-333 diet. In addition, expression of cytochrome p450 4a14 and acyl-CoA oxidase was normalized, and the percentages of positive cells for 8-hydroxy-2'-deoxyguanosine, 4-hydroxy-2-nonenal and proliferating cell nuclear antigen were decreased. Furthermore, expression of beta-catenin and cyclin D1 was also depressed. These data suggest that NIK-333 suppressed liver tumor in association with repression of oxidative stress.

Retinoids have several biological functions including cell growth, differentiation and apoptosis. In liver, retinoids are known to be associated with regeneration, fibrosis and carcinogenesis. The facts that retinoid droplets in stellate cells are lost with progression of liver disease and that effectiveness of an acyclic retinoid on second primary liver cancer suggest the importance of liver as a target organ of retinoids. Our recent work has indicated that retinoids have antioxidant effects in association with regulation of fatty acid metabolism. In this review article, we discussed the important function of retinoids in liver, mainly from molecular aspects.

Background The developmental changes of Ni2+-sensitivity to automaticity of Nkx2.5-positive cells derived from mouse embryonic stem cell have been identified, suggesting developmental regulation of expressing Ni2+-sensitive T-type Ca2+ channel, although the mechanism of the change has not been fully studied. Methods and Results Transcripts of Cav3.2, Cav3.1 and Cav1.2 genes of beating Nkx2.5-positive cells, which encode the Ni2+-sensitive T-type Ca2+ channel, Ni2+-insensitive T-type Ca2+ channel, and L-type Ca2+ channel, respectively, were investigated by real-time reverse-transcriptase-polymerase chain reaction, and the current density of each channel was measured by patch-clamp techniques at the early and late stages of differentiation. The expression of the Cav3.2 transcript predominated in the early stage whereas those of Cav3.1 and Cav1.2 transcripts were upregulated in the late stage, which was consistent with the change in each current density, suggesting the expression of channel proteins is largely determined at the transcriptional level. Conclusion The results indicate that the mechanism of change of Ni2+-sensitivity is partly, if not completely, the subtype switch of T-type Ca2+ channel from Cav3.2 to Cav3.1 at the transcriptional level, and that the expression of the L-type Ca2+ channel might have an attenuating effect on Ni2+-sensitivity to automaticity in the late stage of differentiation. (Circ J 2005; 69: 1284 - 1289)

Background Azimilide reportedly blocks Na+ channels, although its mechanism remains unclear. Methods and Results The kinetic properties of the azimilide block of the wild-type human Na+ channels (WT: hH1) and mutant ΔKPQ Na+ channels (ΔKPQ) expressed in COS7 cells were investigated using the whole-cell patch clamp technique and a Markovian state model. Azimilide induced tonic block of WT currents by shifting the h∞ curve in the hyperpolarizing direction and caused phasic block of WT currents with intermediate recovery time constant. The peak and steady-state ΔKPQ currents were blocked by azimilide, although with only a slight shift in the h∞ curve. The phasic block of peak and steady-state ΔKPQ currents by azimilide was significantly larger than the blocking of the peak WT current. The affinity of azimilide predicted by a Markovian state model was higher for both the activated state (KdA =1.4 μmol/L), and the inactivated state (KdI =1.4 μmol/L), of WT Na+ channels than that for the resting state (KdR =102.6 μmol/L). Conclusions These experimental and simulation studies suggest that azimilide blocks the human cardiac Na+ channel in both the activated and inactivated states. (Circ J 2004; 68: 703 - 711)

The long-term goal of our study is to explore how Epstein-Barr virus (EBV) associates with the pathogenesis of gastric carcinoma. The first goal is to establish EBV-positive epithelial cell lines from EBV-infected gastric carcinoma tissues and to characterize the cell lines and EBV infection in the cells. EBV, a ubiquitous human herpesvirus with oncogenic potential, is predominantly associated with the infection of two target tissues in vivo: (1) B lymphocytes, where the infection is largely nonproductive, and (2) the epithelium, in which virus replication occurs (Rickinson and Kieff 1996). Both target tissues are susceptible to EBV-associated malignant change, leading to tumors of B-cell origin, such as Burkitt’s lym-phoma, or of epithelial-cell origin, such as nasopharyngeal carcinoma (NPC) (Pathmanathan et al. 1995). Recently, EBV has also emerged as an etiologic agent implicated in gastric carcinoma (Min et al. 1991; Shibata et al. 1991; Shibata and Weiss 1992; Tokunaga et al. 1993a,b; Fukayama et al. 1994; Imai et al. 1994; Ohfuji et al. 1996; Iwasaki et al. 1998). EBV has been found in most cases of rare gastric lymphoepithelioma-like carcinomas (Min et al. 1991; Shibata et al. 1991; Ohfuji et al. 1996) and a small but significant proportion of common gastric adenocarcinomas (Shibata and Weiss 1992; Tokunaga et al. 1993a,b). EBV infection was found in approximately 7% of Japanese gastric carcinomas (Tokunaga et al. 1993a,b; Fukayama et al. 1994; Imai et al. 1994). The world distribution of gastric carcinomas with EBV infection is shown from 4% to 18% of total gastric carcinoma in different countries (Tashiro et al. 1998)

It is well known that Epstein-Barr virus (EBV) is excreted from oral regions in the patients with infectious mononucleosis. We analyzed the prevalence of EBV in saliva and throat washings from healthy people in Japan by the polymerase chain reaction assay. EBV DNA was detected in 43 (90%) of the 48 throat washings from healthy adults (21 to 57 years old) and in 35 (38%) of the 93 salivas from healthy children (0 to 6 years old). The percentages of the EBV DNA-positive ratio in salivas increased in proportion relative to the increase of the children's ages. EBV type 1 was predominant and was detected in 86 and 94% of adults and children, respectively. Umbilical cord lymphocytes were transformed by some throat washings from EBV seropositive donors. EBV DNA was detected in throat washings from two healthy adults whose EBV antibody was not detected. In both cases, higher amounts of EBV DNA were detected in their peripheral blood mononuclear cells than in those of other, EBV antibody-positive donors. These results demonstrated the incidence of EBV excretion in oral regions of healthy individuals in Japan and defined a novel type of EBV infection in healthy adults.

Truncated and full-length epidermal growth factor (EGF) receptors are produced in rat liver cells. The truncated EGF receptor mRNA is almost identical to the full-length EGF receptor mRNA except for the lack of a 3' region of the full-length receptor mRNA. To understand the stability of rat EGF receptor mRNAs, we analyzed the expression of EGF receptor mRNAs in the hepatoma cell line, AH66 and liver cells. Ten, 7 and 5 kb full-length and 2.7 kb truncated EGF receptor mRNAs were detected in both of them. The half-lives of the 10 and 2.7 kb EGF receptor mRNAs were determined in AH66 cells using a transcriptional inhibitor, 5,6-dichlororibofuranosylbenzimidazole. The half-lives of the 10 and 2.7 kb mRNAs were 1.2 and 11 h, respectively. These results indicated that the truncated mRNA is 4 times more stable than the full-length mRNA in rat cells. As for the stability, the role of a sequence of 3'-untranslated region of the EGF receptor mRNA was discussed.

Aims; It has been reported that hepatitis C virus (HCV) core expression causes hepatocellular carcinoma (HCC) in transgenic mice by reactive oxygen species (ROS). On the other hand, we have reported that loss of signals of retinoic acid (RA), a metabolite of vitamin A, leads to steatohepatitis, eventually to HCC in the receptor for RA (RARα)-knocked-down mice. We have demonstrated that the mechanism by which loss of RA signal causes HCC is mediated by increased oxidative stress via impaired metabolisms of fatty acids and iron. In turn, the effect of oxidative stress on RA signals remains to be clarified. The present study aims to explore the effect of oxidative stress and HCV core expression on RA signals in the context of hepatocarcinogenesis. Methods; The first parts of experiments were done using hydrogen peroxide as the source of ROS Human HCC cell lines HuH7, HLF, HepG2, Hep3B, Li7, Alexander and HuH6 were used. The expression level of RARα and activation of MAPKs such as ERK, JNK and p38, and RA signal by DR5-reporter assay were examined with 0.1∼1μM hydrogen peroxide. Anisomycin, an activator of JNK signal, SP600125, an inhibitor of JNK, and constitutive active MKK7-JNK1 was explored on level of RARα and activation of MAPKs. Phosphorylation and ubiquitination of mutant RARα, of which three phosphorylation sites by JNK are changed from serines to alanines, were examined. The second parts of experiments were performed using tetracycline-inducible HCV core system (Tet-On system), which is constructed in HuH7 cells. The intensity of RA signal after induction of HCV core protein was measured by DR5-reporter assay. The expression Sp101b, which is induced by RA and an adaptive protein of HCV core, was examined on expression and cellular translocation. The level of SRC-3, a coactivator of RAR/RXR, and p38 MAPK, an upstream signal of SRC-3 and a downstream signal of oxidative stress, were examined. The cellular antioxidative ability and hydroxyl radical formation were measured during this process. Results; In the first parts experiment using hydrogen peroxide, oxidative stress suppressed RA signal through degradation of RARα. The degradation of RARα was mediated by its phosphorylation by JNK and followed by ubiquitination. In the second parts of experiments, expression of HCV core protein reached its maximum at 72 h at 5 μg/ml of Tet. RA signal increased at 36 h, however then decreased at 48 h and 72 h. The decreased RA signal was reverted by N-acetyl cystein (NAC), an antioxidative protein. The cellular antioxidative ability was decreased at 24h, and hydroxyl radical formation was increased at 48 h. Activation of p38 and SRC-3 was observed after induction of HCV core expression, however it was inhibited by NAC. Conclusions; The data of the present study suggest that RA plays an important role in preventive effect of hepatitis C virus- and oxidative stress-related hepatocarcinogenesis Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5723.

Background & Aims We previously reported that impaired retinoid signaling in the liver causes steatohepatitis and hepatocellular carcinoma. Recently, oxidative stress induced by hepatic iron overload has emerged as an important factor for the progression of liver disease in patients with chronic hepatitis C, alcoholic liver disease, and nonalcoholic steatohepatitis. In this study, the relationship between retinoid signaling and iron metabolism in the liver was investigated. Methods The effect of retinoids on the iron metabolism was examined in HuH7 cells treated with all- trans retinoic acid and acyclic retinoid NIK-333. In in vivo experiments, we used the mice expressing the dominant negative form of retinoic acid receptor α gene under the control of albumin enhancer/promoter (RAR-E Tg) and iron-overloaded wild mice fed with retinoid-deficient and retinoid-excess diets. Results Hepatic iron accumulation and increased expression of hemojuvelin were observed in RAR-E Tg mouse liver. Retinoid treatment significantly suppressed expression of hemojuvelin and mildly suppressed expression of transferrin receptor type 2 and hepcidin, accompanied by decreased hepatic iron content and iron-induced oxidative stress in vitro and in vivo. Overexpression of hemojuvelin in HuH7 hepatoma cells led to a significant increase in cellular iron content. Conclusions Our results suggest that retinoids are involved in hepatic iron metabolism through transcriptional regulation of hemojuvelin. This study demonstrated a novel functional role of retinoids in preventing iron-induced oxidative stress in the liver.

Epidermal growth factor (EGF) receptor mRNA expression in livers of newborn, adult control and partially hepatectomized rats 1 and 3 days post-operation was examined by Northern blot analysis. A human EGF receptor cDNA (P 64-1, Ullrich et al., 1984) was hybridized to RNA species of 10, 6 and 3 kb. These mRNAs were profoundly decreased in livers from newborn rats and relatively decreased in regenerating rat livers. beta-Actin mRNA in these tissues showed a tendency to increase over that of the adult controls.

Binding sites for mouse epidermal growth factor (EGF) were observed in purified nuclear fraction from rat liver. The binding data yielded a curvilinear Scatchard plot which was decomposed into two binding components with different binding affinities. The binding component with higher affinity (Kd approximately 10(-10) M) represented approximately 15% of the total binding, while the high affinity binding sites were less than 5% of the total in the microsomes. The ligand-dependent autophosphorylation, one of the characteristic features of EGF receptor in the plasma membrane, was not observed at the site of the receptor (170 KDa) in the nuclear fraction. The binding characteristics for EGF fluctuated during the course of liver regeneration after partial hepatectomy; the binding capacity in the nuclear fraction increased in contrast to the decrease in the microsomes. However, the binding sites in the nuclear fraction obtained in the early period after partial hepatectomy consisted only of low affinity ones.

To elucidate the molecular mechanism of rat thymus involution induced by administration of glucocorticoid, we screened a cDNA library for polysomal poly (A) +RNA from adrenalectomized rat thymi by a differential colony hybridization method. Labeled cDNAs for mRNAs isolated from thymi of adrenalectomized rats and rats receiving dexamethasone (Dex) treatment were used as probes. Eight cDNA clones for mRNAs which had a diminished response to administration of Dex were isolated. The relative concentration of cloned mRNAs in the thymic polysomal RNA was significantly decreased 3 h after hormone administration, while changes in the nuclear RNA were not significant after Dex administration. One of the selected cDNA clones designated pRTGR-8 corresponded to mRNA of about 3, 000 nucleotides, and a nuclear run-off transcription assay indicated that the rate of transcription of pRTGR-8 RNA was repressed by Dex administration. The cloned cDNAs obtained in this experiment may provide useful probes for studying the negative regulation mechanism of gene expression by glucocorticoids.

The nucleotide sequence of Dictyostelium discoideum rDNA extending over almost the entire transcribed region and a part of the 5' non-transcribed spacer region has been determined. Computer analysis revealed that there were several conserved sequences in the 17S, 5.8S and 26S coding regions when compared with the sequences at analogous positions in some eukaryotic rRNA genes. The data also showed that the D. discoideum rDNA contains several extra sequences, which have not been found in other eukaryotes' rDNAs , near the 3' terminus of the 17S coding region and the 5' terminus of the 26S coding region.