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The viral genome quasispecies composition of hepatitis C virus (HCV) could have important implications to viral pathogenesis and resistance to anti-viral treatment. The purpose of the present study was to profile the HCV RNA quasispecies. We developed a strategy to determine the full-length HCV genome sequences co-existing within a single patient serum by using next-generation sequencing technologies. The isolated viral clones were divided into the groups that can be distinguished by core amino acid 70 substitution. Subsequently, we determined HCV full-length genome sequences of three independent dominant species co-existing in the sequential serum with a 7-year interval. From phylogenetic analysis, these dominant species evolved independently. Our study demonstrated that multiple dominant species co-existed in patient sera and evolved independently.

Background and purpose Locoregional recurrence after carbon-ion radiotherapy (CIRT) for primary head and neck malignancies, such as malignant mucosal melanoma, adenoid cystic carcinoma, and sarcoma, occurs occasionally. However, the treatment options are limited. We report on the toxicity and efficacy of re-irradiation using carbon ions for recurrent head and neck malignancies after CIRT. Materials and methods Data of 48 patients with recurrent head and neck malignancies treated with re-irradiation with CIRT at our institution (2007–2016) were retrospectively analyzed. Twenty-one patients (43.8%) had malignant mucosal melanoma, 17 (35.4%) had adenoid cystic carcinoma, six (12.5%) had bone and soft tissue sarcomas, and four patients (8.3%) had other disease types. Tumor recurrences at re-irradiation were located in the paranasal cavity (n = 18, 37.5%), nasal cavity (n = 9, 18.8%), nasopharynx (n = 4, 8.3%), orbit (n = 3, 6.3%), cavernous sinus (n = 3, 6.3%), and at other sites (n = 11, 22.9%). The median dose of initial CIRT and that at re-irradiation were 57.6 Gy and 54.0 Gy (relative biological effectiveness [RBE]), respectively. None of the patients received concurrent chemotherapy. Results The median follow-up period after re-irradiation was 27.1 months. Five patients (10.4%) developed Grade 3 acute toxicities and 18 (37.5%) developed Grade ≥3 late toxicities, including Grade 5 central nervous system necrosis in one patient. The 2-year local control, locoregional control, progression-free survival, and overall survival rates were 40.5, 33.5%, 29.4%, and 59.6%, respectively. Conclusion Re-irradiation using carbon ions may be a reasonable treatment option with tolerable toxicity for patients with recurrent head and neck malignancies after CIRT.

Summary There is risk of mandibular osteoradionecrosis following external radiotherapy to tumors at the base of the tongue. To date, no report evaluates sparing of the mandibular bone offered by use of spacers. Here, we introduce and evaluate a custom-made mouthpiece employing a spacer for reduction of osteonecrosis following carbon-ion radiotherapy of tongue base tumors.

The effect of skeletal muscle fat deposition on the prognosis of patients with chronic liver disease remains unclear. Skeletal muscle fat deposition can be estimated by attenuation of skeletal muscle in Hounsfield units (HU) on computed tomography (CT). The aim of this retrospective cohort study was to investigate the association between skeletal muscle fat deposition assessed by skeletal muscle attenuation (SMA), and hepatocellular carcinoma (HCC).We enrolled 288 patients with chronic liver disease (139 men, 149 women; mean age 67.5 ± 10.4 y; hepatitis C virus, 239; hepatitis B virus, 17; without viral infection, 32; chronic hepatitis, 227; and cirrhosis, 61) who underwent liver biopsy and CT scanning between January 2013 and February 2017. The patients were divided into two groups based on SMA levels, with the cutoff value of 31 HU. We analyzed the effect of SMA on HCC development.During the study follow-up period (median, 2.50 y; range, 0.5-4.7 y), HCC was identified in 19 patients (7%). The cumulative incidence of HCC in patients with lower SMA (31 HU) was significantly higher than in patients with SMA ≥31 HU (P = 0.007). Cox proportional hazards regression analysis confirmed cirrhosis (hazard ratio [HR], 6.626; 95% confidence interval [CI], 2.57-17.12; P 0.001) and lower SMA (HR, 3.502; 95% CI, 1.25-9.83; P = 0.017) as significant independent factors associated with HCC development in patients with chronic liver disease.Patients with cirrhosis and skeletal muscle fat deposition assessed by SMA had a higher risk for developing HCC.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that mostly occurs in the elderly. Merkel cell polyomavirus (MCPyV) is detected in approximately 80% of MCCs and is associated with carcinogenesis. Hedgehog signaling pathway plays a role in human embryogenesis and organogenesis. In addition, reactivation of this pathway later in life can cause tumors. Twenty-nineMCPyV-positive and 21 MCPyV-negative MCCs were immunohistochemically stained with primary antibodies for hedgehog signaling (SHH, IHH, PTCH1, SMO, GLI1, GLI2, and GLI3) and evaluated using H-score. Polymerase chain reaction and sequence analysis for SHH and GLI1 exons were also performed. Expression of SHH was higher in MCPyV-positive MCCs than in MCPyV-negative MCCs (P.001). Higher expression of GLI1, MCPyV infection, male sex, and Japanese ethnicity were associated with better overall survival (P=.034, P=.001, P=.042, and P=.036, respectively). Higher expression of SHH and MCPyV infection were associated with improved MCC-specific survival (P=.037 and P=.002, respectively). The mutation analysis of prognosis-related GLI1 and SHH genes in our study revealed a low frequency of mutations in the 10 exons examined, except GLI1 exon 5 (18/22 cases), all having the same silent mutation of c.576GA. Only 2 mutations with amino acid changes were detected in MCPyV-negative MCCs only: 1 missense mutation in GLI1 exon 4 and 1 nonsense mutation in SHH-3B. Expression of SHH and GLI1 may be useful prognostic markers of MCC because increased expression was associated with better prognosis. The high rate of c.576GA silent mutation in GLI1 exon 5 was a feature of MCC.

For successful treatment for nonalcoholic steatohepatitis (NASH), it may be important to treat the individual causative factors. At present, however, there is no established treatment for this disease. Branched-chain amino acids (BCAAs) have been used to treat patients with decompensated cirrhosis.In order to elucidate the mechanisms responsible for the effects of BCAAs on hepatic steatosis and disease progression, we investigated the effects of BCAA supplementation in mice fed a choline-deficient high-fat diet (CDHF), which induces NASH.Male mice were divided into four groups that received (1) choline-sufficient high fat (HF) diet (HF-control), (2) HF plus 2% BCAA in drinking water (HF-BCAA), (3) CDHF diet (CDHF-control), or (4) CDHF-BCAA for 8weeks. We monitored liver injury, hepatic steatosis and cholesterol, gene expression related to lipid metabolism, and hepatic fat accumulation.Serum alanine aminotransferase (ALT) levels and hepatic triglyceride (TG) were significantly elevated in CDHF-control relative to HF-control. Liver histopathology revealed severe steatosis, inflammation, and pericellular fibrosis in CDHF-control, confirming the NASH findings. Serum ALT levels and hepatic TG and lipid droplet areas were significantly lower in CDHF-BCAA than in CDHF-control. Gene expression and protein level of fatty acid synthase (FAS), which catalyzes the final step in fatty acid biosynthesis, was significantly decreased in CDHF-BCAA than in CDHF-control (P0.05). Moreover, hepatic total and free cholesterol of CDHF-BCAA was significantly lower than those of CDHF-control.BCAA can alleviate hepatic steatosis and liver injury associated with NASH by suppressing FAS gene expression and protein levels.

Randomly grain oriented Bi2223 sintered bulks are one of the representative superconducting materials having weak-link problem due to very short coherence length particularly along the c -axis, resulting in poor intergrain J c properties. In our previous studies, sintering and/or post-annealing under moderately reducing atmospheres were found to be effective for improving grain coupling in Bi2223 sintered bulks. Further optimizations of the synthesis process for Bi2223 sintered bulks were attempted in the present study to enhance their intergrain J c . Effects of applied pressure of uniaxial pressing and sintering conditions on microstructure and superconducting properties have been systematically investigated. The best sample showed intergrain J c of 2.0 kA cm −2 at 77 K and 8.2 kA cm −2 at 20 K, while its relative density was low ∼65%. These values are quite high as for a randomly oriented sintered bulk of cuprate superconductors.

Merkel cell carcinoma (MCC) is a clinically aggressive neuroendocrine skin cancer; 80% of the cases are associated with the Merkel cell polyomavirus (MCPyV). We previously reported that MCPyV-negative MCCs have more irregular nuclei with abundant cytoplasm and significantly unfavorable outcomes than do MCPyV-positive MCCs. These results suggest that some cell adhesion or structural stabilization molecules are differently expressed depending on MCPyV infection status. Thus, we investigated the association of prognosis or MCPyV infection status in MCCs with cell adhesion molecule 1 (CADM1)/differentially expressed in adenocarcinoma of the lung protein 1 (DAL-1)/membrane protein, palmitoylated 3 (MPP3) tripartite complex and mal T-cell differentiation protein (MAL) expression, which play important roles in cell adhesion and oncogenesis and are related to cancer outcomes in various malignancies, to elucidate the role of these molecules. We analyzed the pathological and molecular characteristics of 26 MCPyV-positive and 15 MCPyV-negative MCCs. Univariate Cox regression analysis showed that advanced age (hazard ratio [HR], 8.249; P = .007) and high CADM1 expression (HR, 5.214; P = .012) were significantly unfavorable overall survival parameters, whereas MCPyV infection (HR, 0.043, P < .001) and lower MAL expression (HR, 0.273; P = .018) were significantly favorable. On multivariate analysis, only MCPyV infection was significantly favorable for overall survival (HR, 0.04; P = .005). Hypermethylation of CADM1, DAL-1, and MAL promoters was detected in 1 of 18, 15 of 27, and 1 of 13 cases, respectively. Double immunostaining for cytokeratin 20 and CADM1, DAL-1, or MAL showed that nonneoplastic Merkel cells expressed DAL-1 and MAL but not CADM1. This study revealed that MCPyV-negative MCCs significantly expressed higher CADM1 and lower MAL than MCPyV-positive MCCs; these expression levels were markedly related to unfavorable outcomes. These data will give us important insights to develop novel molecular target therapies for MCCs.

Ishikari project constructs two lines. The length of the Line 1 is 500 m, and connects the photovoltaic cell to the internet-data center. The other line is 1 km length, and it is a test facility and called Line 2. The structures of the cable systems are not same to test their performance. The construction was started from 2014 in the field, the Line 1 was completed in May 2015, and it was cooled down and do the current experiment, and warmed up. The Line 2 is almost complete in October 2015. It will be tested in November and December, 2015. In order to reduce the stress of the cable induced by the thermal expansion and contraction, we adopted the way of the helical deformation of the cable. The force of the cable is reduced to 1/3 of an usual cable test. Because the cryogenic pipes are welded in the field and we cannot use the baking of the vacuum chamber of the cryogenic pipe, a new vacuum pumping method was proposed and tested for the cryogenic pipe. Since the straight pipes are used to compose the cryogenic pipe, the pressure drop of the circulation would be 1/100 of the corrugated pipe in the present condition, and it is suitable for longer cable system. The heat leak of the cryogenic pipe is ∼1.4W/m including the cable pipe's and the return pipe's. The heat leak of the current lead is ∼30W/kA in the test bench. Finally the current of 6kA/3 sec and the current of 5kA/15 min were achieved in Line 1. The reduction of heat leak will be a major subject of the longer cable system. The cost of the construction will be almost twice higher than that of the copper and aluminum over-head line with the iron tower in the present Japan. The cost construction of the over-head line is an average value, and depends on the newspaper.

Nonalcoholic steatohepatitis (NASH) patients progress to liver cirrhosis and even hepatocellular carcinoma (HCC). Several lines of evidence indicate that accumulation of lipopolysaccharide (LPS) and disruption of gut microbiota play contributory roles in HCC. Moreover, in a dextran sodium sulfate (DSS)-induced colitis model in mice, a high-fat diet increases portal LPS level and promotes hepatic inflammation and fibrosis. However, this diet-induced NASH model requires at least 50 weeks for carcinogenesis. In this study, we sought to determine whether increased intestinal permeability would aggravate liver inflammation and fibrosis and accelerate tumorigenesis in a diet-induced NASH model. Mice were fed a choline-deficient high-fat (CDHF) diet for 4 or 12 weeks. The DSS group was fed CDHF and intermittently received 1% DSS in the drinking water. Exposure to DSS promoted mucosal changes such as crypt loss and increased the number of inflammatory cells in the colon. In the DSS group, portal LPS levels were elevated at 4 weeks, and the proportions of Clostridium cluster XI in the fecal microbiota were elevated. In addition, levels of serum transaminase, number of lobular inflammatory cells, F4/80 staining-positive area, and levels of inflammatory cytokines were all elevated in the DSS group. Liver histology in the DSS group revealed severe fibrosis at 12 weeks. Liver tumors were detected in the DSS group at 12 weeks, but not in the other groups. Thus, DSS administration promoted liver tumors in a CDHF diet-induced NASH mouse over the short term, suggesting that the induction of intestinal inflammation and gut disruption of microbiota in NASH promote hepatic tumorigenesis.

The hepatoprotective action of ursodeoxycholic acid (UDCA) was previously suggested to be partially dependent on its antioxidative effect. Doxorubicin (DOX) and reactive oxygen species have also been implicated in the overexpression of P-glycoprotein (P-gp), which is encoded by the MDR1 gene and causes antitumor multidrug resistance. In the present study, we assessed the effects of UDCA on the expression of MDR1 mRNA, P-gp, and intracellular reactive oxygen species levels in DOX-treated HepG2 cells and compared them to those of other bile acids. DOX-induced increases in reactive oxygen species levels and the expression of MDR1 mRNA were inhibited by N-acetylcysteine, an antioxidant, and the DOX-induced increase in reactive oxygen species levels and DOX-induced overexpression of MDR1 mRNA and P-gp were inhibited by UDCA. Cells treated with UDCA showed improved rhodamine 123 uptake, which was decreased in cells treated with DOX alone. Moreover, cells exposed to DOX for 24h combined with UDCA accumulated more DOX than that of cells treated with DOX alone. Thus, UDCA may have inhibited the overexpression of P-gp by suppressing DOX-induced reactive oxygen species production. Chenodeoxycholic acid (CDCA) also exhibited these effects, whereas deoxycholic acid and litocholic acid were ineffective. In conclusion, UDCA and CDCA had an inhibitory effect on the induction of P-gp expression and reactive oxygen species by DOX in HepG2 cells. The administration of UDCA may be beneficial due to its ability to prevent the overexpression of reactive oxygen species and acquisition of multidrug resistance in hepatocellular carcinoma cells.

The implant stability quotient (ISQ) has been widely evaluated in clinical studies and animal experiments. However, accurate measurement is often difficult in animal models. In such cases, measurement of ISQ in bone is needed after formalin fixation. However, it is not yet clear how such fixation influences the measurement. The purpose of this study was to investigate the influence of formalin fixation on ISQ and the mechanical characteristics of bone. Fourteen tibias were harvested from rabbits; the samples from the left (length 60mm, control group) were soaked in saline and the samples from the right (length 60mm, fixation group) were fixed in 10% neutral-buffered formalin at 4°C for 4h. Three-point bending tests were done at 5mm/min to measure the maximum load and total absorbed energy. Twelve titanium implants (Brånemark System(®) Mk-III TiUnite, Nobel Biocare, Sweden) were placed into the edentulous molar site of the mandibles of 2 dogs and the ISQ was measured by Osstell(®) (control group) 3 months later. The implants involved in the bone block were then fixed for 4h (fixation group) and the ISQ measured. The maximum load values did not differ significantly between the control and fixation groups. Total absorbed energy was significantly higher in the control group than in the fixation group. ISQ did not differ significantly between the groups. These results suggest that formalin fixation of bone might affect some of the mechanical characteristics of bone, but not its ISQ.

A Short Time Exposure (STE) test is a cytotoxicity test that uses SIRC cells (rabbit corneal cell line) to assess eye irritation potency following a 5-min chemical exposure. This second-phase validation study assessed the predictive capacity of the STE test using 40 coded test substances at three laboratories. A Validation Management Team (VMT) then evaluated the predictivity of the STE test for United Nation (UN) Globally Harmonized System (GHS) categories using 63 test substances including the results of the first-phase validation study. The STE test can assess not only the severe or corrosive ocular irritants (corresponding to the UN GHS Category 1) but also non-irritant (corresponding to UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. The predictivity by STE test, however, was insufficient for identification of UN GHS categories (Category 1, Category 2, or Non Category). These results suggest that the STE test can be recommended as an initial step in a top-down approach to identification of severe irritants and test substances that require classification for eye irritation (UN GHS Category 1) as well as an initial step in a bottom-up approach to identification of test substances that do not require classification for eye irritation (UN GHS Non Category) from other toxicity classes, especially for limited types of test substances. On the other hand, the STE test is not considered adequate for the identification of mild or moderate irritants (i.e., UN GHS Categories 2A and 2B) and severe irritants (UN GHS Category 1).

Merkel cell polyomavirus infects up to 80% of patients with Merkel cell carcinoma. Combined Merkel cell carcinoma and cutaneous tumors occur occasionally. Previous reports have suggested that Merkel cell polyomavirus is absent from combined Merkel cell carcinoma and squamous cell carcinomas. This is the first report that Merkel cell polyomavirus infected in both lesions of a combined Merkel cell carcinoma and basal cell carcinoma. A 92-year-old Japanese man presented with a right thigh small subcutaneous mass. Histologic examination revealed a combined tumor with Merkel cell carcinoma and basal cell carcinoma with ductal differentiation. Both tumors and intermingled Merkel cells in basal cell carcinoma expressed Merkel cell polyomavirus large T antigen, and 17 and 240 copies of Merkel cell polyomavirus/cell were detected in the microdissected Merkel cell carcinoma and basal cell carcinoma specimens, respectively. Mutation analysis of Merkel cell polyomavirus large T antigen revealed a novel truncating mutation in Merkel cell carcinoma and a similar but different mutation in the basal cell carcinoma. These results suggest that each was infected by a different Merkel cell polyomavirus subclone derived from a single Merkel cell polyomavirus.

Hepcidin is an iron-regulatory hepatic peptide hormone encoded by the HAMP gene that downregulates iron export from enterocytes and macrophages into the blood plasma. In this study, we identified a novel mutation in the HAMP gene of a 58-year-old Japanese male patient with hemochromatosis. By direct sequencing of the five hereditary hemochromatosis-related genes, HFE, HAMP, HJV, TFR2, and SLC40A1, the previously unreported p.R75X mutation was identified, and the patient was found to be homozygous for the mutation. No other potentially pathogenic mutations were detected. In an LC-MS/MS analysis, hepcidin molecules were not detected in the patient's serum or urine. These results indicate that the p.R75X mutation causes iron overload by impairing the hepcidin system.

Enhancement of J c performance of Bi(Pb)2223 bulks was attempted by sintering under controlled over pressures (CT-OP) and c -axis grain orientation. Relative density of bulks increased up to 93% with an increase of total gas pressure during the sintering. In addition, improvement of grain coupling by CT-OP sintering was indicated from normal state resistivity. Reflecting these facts, the bulks sintered by the CT-OP method showed higher J c than the bulks sintered under ambient pressure. On the other hand, magnetic grain alignment technique was adopted to achieve c -axis grain orientation. The c -axis oriented bulks showed higher J c than randomly oriented bulks. The observed J c of 2.3 x 104 Acm-2 (20 K, 1 kOe) was very high value as for Bi(Pb)2223 sintered bulks.

Short Time Exposure (STE) test is an easy in vitro eye irritation test that assesses cytotoxicity in SIRC cells (rabbit corneal cell line) following a 5 min dose treatment. To assess intra-laboratory reproducibility, medium control, three vehicles (saline, saline containing 5% (w/w) dimethyl sulfoxide, and mineral oil) and three standard chemicals (sodium lauryl sulfate, calcium thioglycolate, and Tween 80) were evaluated. Assessments were repeated 30 times for vehicles and 18 times for standard chemicals; resulting in almost the same cell viability and a low coefficient of variation value. In addition, the STE eye irritation rankings of three standard chemicals, as calculated on the cell viabilities in 5% and 0.05% solutions were in agreement in all tests. Based on these results, high intra-laboratory reproducibility was confirmed. In addition, the irritation category (irritant and non-irritant) was evaluated for 109 chemicals with STE test, globally harmonized system (GHS) classification, and European Union (EU) classification. The results of the evaluation found the STE classification to have an accuracy with GHS classification of 87% and with EU classification of 83%, which confirmed the excellent correspondence. The correspondence of STE rankings (1, 2, and 3) based on the prediction model by STE test with the eye irritation rankings by GHS (non-irritant, categories 2 and 1) and EU (non-irritant, R36, and R41) was 76% and 71%, respectively. Based on the above results, STE test was considered to be a promising alternative method for assessing eye irritation that has high intra-laboratory reproducibility as well as an excellent predictability of eye irritation.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) kinase (BDK) is responsible for the regulation of BCKDH complex, which is the rate-limiting enzyme in the catabolism of branched-chain amino acids (BCAAs). In the present study, we investigated the expression and activity of hepatic BDK in spontaneous type 2 diabetes using hyperinsulinemic Zucker diabetic fatty rats aged 9weeks and hyperglycemic, but not hyperinsulinemic rats aged 18weeks. The abundance of hepatic BDK mRNA and total BDK protein did not correlate with changes in serum insulin concentrations. On the other hand, the amount of BDK bound to the complex and its kinase activity were correlated with alterations in serum insulin levels, suggesting that hyperinsulinemia upregulates hepatic BDK. The activity of BDK inversely corresponded with the BCKDH complex activity, which was suppressed in hyperinsulinemic rats. These results suggest that insulin regulates BCAA catabolism in type 2 diabetic rats by modulating the hepatic BDK activity.

Topical application of ceramides is reported to improve the structure and texture of the skin. Synthetic pseudo-cermaide, SLE66 has been shown to reduce dryness/scaling/itching of human skin. Although efficacy of topically applied ceramides and their analogs has been investigated to some extent, safety information is scarce. The objective of the present investigation was to evaluate potential adverse effects of SLE66. The oral LD(50) of SLE66 in rats and mice was >5000 mg/kg, while dermal LD(50) in rats was >2000 mg/kg. In animal and human studies, SLE66 did not cause skin irritation or sensitization. SLE66 does not possess phototoxicity or photosensitization potentials. Instillation of SLE66 into rabbit eye elicited transient conjunctival irritation. In 28 day repeat-dose studies, administration of SLE66 via gavage (daily) or by dermal application (five days/week) to Sprague Dawley rats at levels up to 1000 mg/kg/day did not cause mortality or morbidity. Compared to the controls, the clinical condition of the animals, body weights, feed consumption, hematology, clinical chemistry, organ weights, and gross necropsy findings were unaffected by oral or dermal administration of SLE66. The no-observed-adverse-effect level (NOAEL) for systemic toxicity following oral or dermal administration of SLE66 was 1000 mg/kg/day (the highest level tested).

A synthetic pseudo-ceramide (SLE66) has been shown to improve dryness/itching of human skin. The objective of this study was to investigate metabolism, genotoxicity and cytotoxicity of SLE66. Pharmacokinetic profile of [14C]-SLE66 was investigated following oral or transdermal administration to Sprague-Dawley rats. The plasma radioactivity following oral administration (1000 mg SLE66/kg) reached maximum (Cmax--39.77 µg eq/ml) at 4 h and then declined with apparent elimination half-life of 23.6 h. Following transdermal application of [14C]-SLE66 (1000 mg/kg) or as prescription formulation (8%; 160 mg/kg) to normal skin-rats, no radioactivity was noted in plasma, while in damaged skin-rats, trace amount of plasma radioactivity was detected for up to 8 h. The radioactivity excreted in urine, feces, and expiratory air collected for up to 24 h accounted for 5.7, 84.8, and 0.6%, respectively. In the cytotoxicity experiments, SLE66 did not cause toxicity in human skin model. In the genotoxicity studies, SLE66 was not mutagenic in Ames assay and chromosomal aberration assay. These results suggest that following oral administration a small amount of SLE66 is absorbed and excreted in urine, while no absorption was noted after transdermal application to normal skin and SLE66 does not cause cytotoxicity or genotoxicity.

Ursodeoxycholic acid (UDCA) is widely recognized as an effective compound in the treatment of chronic hepatitis and is known to modulate the redox state of the liver accompanied by an increase of GSH. In the present study, to access the antioxidative effect of UDCA and to clarify the molecular basis of the action on GSH level, we evaluated its effects in HepG2 cells exposed to excessive iron. UDCA inhibited both a decrease in the GSH level and an increase in the reactive oxygen species caused by excessive iron in the cells. UDCA increased the gene expression of the catalytic- and modifier-units of glutamine-cysteine ligase (GCL), which is a key enzyme in GSH synthesis. We further investigated the effect of UDCA on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and obtained results showing that UDCA-induced increase in the GSH level was prevented by LY294002, a PI3K inhibitor. In addition, Western blot analysis of Akt showed that, while the total Akt level remained unchanged, the phosphorylated Akt level was increased by UDCA, and this increase was also prevented by LY294002. Moreover, UDCA promoted the translocation of a transcription factor, nuclear factor-E2-related factor-2 (Nrf2), into the nucleus, and this action was inhibited by LY294002. From these results, it was indicated that UDCA increased the GSH synthesis through an activation of the PI3K/Akt/Nrf2 pathway. This may be a primary mechanism of antioxidative action of UDCA concerned with its therapeutic effectiveness in chronic hepatitis.

Aerosol samples were collected at the summit of Mt. Fuji (3776 m a.s.l., at 35.21N, 138.43E) from July 2001 to August 2002, using a high volume air sampler (ca. 700 L/min) with quartz filters. The concentrations of major ionic species were determined using ion chromatography. The concentrations of Ca 2+ , Na + , Mg 2+ , Cl − and NO 3 − were comparable to those reported for the free troposphere and are considered to be close to the background concentrations of the species. The concentrations of Ca 2+ , Na + , Mg 2+ , Cl − and NO 3 − at the summit were higher in spring, than that of SO 4 2− in spring and summer, and that of NH 4 + in summer than those in the rest of the respective seasons. The equivalent concentrations of SO 4 2− and NH 4 + showed a good 1:1 correlation and their stoichiometry indicates the occurrence of (NH 4 ) 2 SO 4 in summer, autumn and winter. The sources of SO 4 2− and NH 4 + are mainly China, but also the Kanto area especially in summer. However, the 1:1 correlation was not seen in spring. It is inferred that NO 3 − and SO 4 2− ions have replaced carbonate in CaCO 3 in Kosa particles, are then transported from the Asian continent. The present study demonstrated the importance of continuous monitoring of the atmosphere at a high elevation in Japan, especially using consistent protocols, and that the summit of Mt. Fuji is one of the best locations for the monitoring of the atmosphere over the Asian continent as well as the western Pacific Ocean.

The branched-chain alpha-keto acid dehydrogenase (BCKDH) complex is the most important regulatory enzyme in branched-chain amino acid (BCAA) catabolism. We examined the regulation of hepatic BCKDH complex activity in spontaneous type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Zucker diabetic fatty rats. Hepatic BCKDH complex activity in these rats was significantly lower than in corresponding control rats. The amount of BCKDH complex in OLETF rats corresponded to the total activity of the complex. Activity and abundance of the bound form of BCKDH kinase, which is responsible for inactivation of the complex, showed an inverse correlation to BCKDH complex activity in OLETF rats. Dietary supplementation of 5% BCAAs for 10 weeks markedly increased BCKDH complex activity, and decreased the activity and bound form of BCKDH kinase in the rats. These results suggest that BCAA catabolism in type 2 diabetes is downregulated and enhanced by BCAA supplementation.

Background & Aims: Hepatitis C virus (HCV) infects millions of people worldwide. Therapy is limited, and treatment does not produce a sustained response in the majority of patients. Development of new agents has been hampered by the lack of a convenient animal model. The aim of this study was to determine whether an immunocompetent rat, tolerized and transplanted with a human hepatoma cell line (Huh 7 cells), could be used to sustain an HCV infection. Methods: Fetal rats were tolerized in utero with 10 5 Huh 7 cells. One day after birth, rats were transplanted with 5 × 10 6 Huh 7 cells and, a week later, inoculated with HCV, genotype 1. Results: In tolerized, transplanted, and HCV-infected rats, Huh 7 cells were found in the liver, and HCV viral replication was detected by the presence of negative strand HCV RNA. HCV levels in serum were measured at 11,000 copies/mL at week 4, peaked at 22,500 copies/mL by week 12. In tolerized, transplanted, inoculated rats, but not controls, serum alanine aminotransferase (ALT) values increased to 60 IU/L by week 4 and reached a peak of approximately 120 IU/L by week 13. Histology showed foci of mononuclear infiltrates in portal and central regions. Conclusions: HCV-inoculated immunocompetent rats tolerized and transplanted with Huh 7 cells support HCV gene expression, viral replication, and develop biochemical and histologic evidence of hepatitis.

Background Although local recurrence of advanced esophageal cancer is frequent after definitive chemoradiotherapy (CRT), the clinical benefit of salvage esophagectomy has not been elucidated. Methods We reviewed 27 patients with squamous-cell cancer who underwent esophagectomy after definitive CRT (≥50 Gy) (salvage group) and 28 patients who underwent planned esophagectomy after neoadjuvant CRT (30 to 45 Gy) (neoadjuvant group). Results The preoperative albumin level and vital capacity were significantly lower in the salvage group than in the neoadjuvant group. Two patients (7.4%) from the salvage group who underwent extended esophagectomy with three-field lymphadenectomy died of postoperative complications, but no deaths occurred after less-invasive surgery. There was no difference of overall postoperative survival between the salvage and neoadjuvant groups. Conclusions The outcome of salvage esophagectomy after definitive CRT was similar to that of planned esophagectomy after neoadjuvant CRT. Less-invasive procedures might be better for salvage esophagectomy because of the high operative risk.

We demonstrated that angiotensin II (Ang II, 10-1000 nM) induced proliferation of cultured rabbit gingival fibroblasts in a concentration-dependent manner. The Ang II-induced proliferation was inhibited by CV-11974 (AT1 antagonist; 1 microM) and saralasin (AT1/AT2 antagonist; 1 microM), but not by PD123,319 (AT2 antagonist; 1 microM), suggesting that Ang II-induced proliferation was mediated via AT1 receptors present in and/or on gingival fibroblasts. The results of Western blot analysis indicated the presence of AT1 and AT2 receptors in/on the fibroblasts. In a subsequent radioligand binding assay, the binding of [3H]Ang II to the fibroblasts was specific and saturable with both high- and low-affinity sites. Competition binding experiments indicated that Ang II completely displaced [3H]Ang II binding, and CV-11974 and PD123,319 maximally displaced up to approximately 63% and 37% of the total binding, respectively. Ang II and CV-11974 completely displaced the [3H]DuP753 binding but PD123,319 did not, indicating a single population of binding site. These findings demonstrate that gingival fibroblasts contain both AT1 and AT2 receptor subtypes for Ang II, and support that Ang II stimulation of AT1 receptors results in proliferation of the fibroblasts.

Comment on ‘Cytokeratin 20-negative Merkel cell carcinoma is infrequently associated with the Merkel cell polyomavirus’

Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.

An aerosol observation campaign was conducted at the summit of Mt. Fuji (at an altitude of 3776 m), which is located 170 km northwest of Miyakejima. Individual aerosol particles were collected with an electrostatic aerosol sampler and the number–size distribution and composition were examined by electron microscopy. Number–size distributions were measured with the combination of a diffusion battery (DB) and a condensation particle counter. Sulfate and sulfur dioxide concentrations were also determined. During the first half of the campaign (13–15 September 2000), high number fractions of sulfuric-acid containing particles with a mode radius around 0.06 μm were observed in the radius range of 0.02–0.2 μm coincident with a humid maritime air mass originated from the south. Sulfate and sulfur dioxide also showed high concentrations in this period. These results suggested that the volcanic plumes of Miyakejima were transported up to the summit of Mt. Fuji. Number–size distributions determined by electron microscope were consistent with those derived using the DB. During the later half of the experiment (18–20 September), low fractions of sulfuric-acid containing particles represented only a small fraction of the total particle number, coincident with a dry continental air mass originating from the west.

Epstein-Barr virus-associated hemophagocytic syndrome (EBV-AHS) is often associated with fatal infectious mononucleosis or T-cell lymphoproliferative diseases (LPD). To elucidate the true nature of fatal LPD observed in Herpesvirus papio (HVP)-induced rabbit hemophagocytosis, reactive or neoplastic, we analyzed sequential development of HVP-induced rabbit LPD and their cell lines. All of the seven Japanese White rabbits inoculated intravenously with HVP died of fatal LPD 18 to 27 days after inoculation. LPD was also accompanied by hemophagocytic syndrome (HPS) in five of these seven rabbits. Sequential autopsy revealed splenomegaly and swollen lymph nodes, often accompanied by bleeding, which developed in the last week. Atypical lymphoid cells infiltrated many organs with a “starry sky” pattern, frequently involving the spleen, lymph nodes, and liver. HVP-small RNA-1 expression in these lymphoid cells was clearly demonstrated by a newly developed in situ hybridization (ISH) system. HVP-ISH of immunomagnetically purified lymphoid cells from spleen or lymph nodes revealed HVP-EBER1+ cells in each CD4+, CD8+, or CD79a+ fraction. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. HVP-DNA was detected in the tissues and peripheral blood from the infected rabbits by PCR or Southern blot analysis. Clonality analysis of HVP-induced LPD by Southern blotting with TCR gene probe revealed polyclonal bands, suggesting polyclonal proliferation. Six IL-2-dependent rabbit T-cell lines were established from transplanted scid mouse tumors from LPD. These showed latency type I/II HVP infection and had normal karyotypes except for one line, and three of them showed tumorigenicity in nude mice. These data suggest that HVP-induced fatal LPD in rabbits is reactive polyclonally in nature.

AC losses were estimated on large current capacity cable conductors on the assumption that current was distributed to each layer uniformly. The estimation clarified wire characteristics and structure for a required large capacity and small loss conductor. From the point of those researches on conductor design, the honeycomb partition structure was chosen. We adapted evacuated extrusion method to making ceramic partition and found 31 A of critical current and 3800 A/cm2 of engineering current density in a calcium carbonate partitioned seven-core wire. AC losses of a strontium carbonate partitioned nineteen-core wire were evaluated. In parallel field, which is a main component in multi-layered cable conductor, some loss reduction was found. The AC losses were also found evident advantages in perpendicular field.

We adopted superconducting parallel conductors to build a 500 kVA transformer cooled by LN 2 . The strands in the parallel conductors are transposed for low ac loss. We have been studying the additional ac loss in parallel conductors, which is induced by the deviation of the transposition points from the optimum ones. It is proportional to the deviation length and the square of the field amplitude, and has Debye-type frequency dependence. However we have not taken into account the effect of the diamagnetic field in the theoretical expressions. It relates with the aspect ratio of a parallel conductor. Hence, in this paper, we investigated the dependence of the additional ac loss on the distance between the strands in a two-strand parallel conductor experimentally.

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.

Annual and seasonal trends were discussed for precipitation chemistry in Japan on the basis of a nationwide monitoring network by Japan Environment Agency. For this analysis, 17 sites, selected from the 23 network sites after evaluation of data completeness for the present purpose, were grouped into four areas in terms of the sea which could characterize each area: the Pacific Ocean, the Japan Sea, the Seto Inland Sea and the East China Sea areas. Major ion concentrations at each site were subjected to least-squares multiple regression analysis, and discussion was principally focused on the area-groups. Each area showed significant (p

The positive effects of physical activity on human bone mass have been well documented in many cross-sectional studies comparing athletes with sedentary controls as well as in longitudinal follow-up. By applying peripheral quantitative computed tomography (pQCT), which has the advantage of measuring volumetric bone mineral density (BMD) and the ability to distinguish among trabecular and cortical components, it was demonstrated that cortical BMD of the dominant arm was not greater than that of the nondominant arm. Cortical drift toward the periosteal direction and an increase in cortical thickness resulted in an improvement of mechanical characteristics of the playing arm’s midradius. An improvement in the mechanical properties of young adult bone in response to long-term exercise was therefore related to geometric adaptation, but not to an increase in BMD. The manner in which the recruitment and function of bone cells are coordinated differs between the growing and the nongrowing skeleton. In the former, modeling is the dominant mode, and in the latter it is remodeling. In the present study, the side-to-side difference of 92 middle-aged female tennis players who initiated training after bone had matured was analyzed by pQCT. The side-to-side difference detected suggested a paradoxical adaptation of the mature radius to unilateral use during tennis playing, and that tennis playing after bone had matured did not stimulate cortical drift in the periosteal direction, unlike that seen in young subjects. Unexpectedly, the cross-sectional areas (periosteal and endocortical area) of the radius were smaller in the dominant arm than in the nondominant arm in the middle-aged female players. The findings suggest that unilateral use of the arm after the third decade of life suppresses age-related changes in bone geometry. (Bone 30:619 – 623; 2002) © 2002 by Elsevier Science Inc. All rights reserved.

Sulfinyldiacetic acid amide ester rac - 1 was efficiently synthesized starting from thiodiacetic acid 4 . Treatment of rac - 1 with Ac 2 O and TMSOTf in CH 2 Cl 2 at −40°C gave chemoselectively amide site α-acetoxy sulfide rac - 2 in a ratio (91:9) of rac - 2 and rac - 3 and in a 90% total yield. Similar treatment of 1 with Ac 2 O and TMSOTf in DMF at room temperature furnished ester site α-acetoxy sulfide rac - 3 in a highly chemoselective manner ( rac - 2 : rac - 3 =3:97) and in a 92% total yield.

When considering treatment for chronic hepatitis B (CHB), it is important to discriminate between patients with persistent low HBV DNA and patients with active hepatitis, who may proceed to cirrhosis. In this study, we sought to identify mutations in patients expected to have persistent low HBV DNA and ultimately exhibit clearance of hepatitis B surface antigen (HBsAg).Serum samples were obtained from 33 CHB genotype C patients, divided based on HBV DNA and alanine aminotransferase (ALT) levels following observation for2 years: Group A (n=10), transient HBV DNA ≥5.0 log copies/mL and ALT ≥120 IU/L; Group B (n=11), persistent HBV DNA5.0 and ALT60; and Group C (n=12), persistent HBV DNA4.0 and ALT30. Full-length HBV sequences were compared among groups. Subsequently, 82 patients with CHB were evaluated for the I97L mutation and the additional mutation P79Q. We compared cumulative incidences of persistent low HBV DNA and HBsAg clearance in patients with or without I97L and P79Q by the Kaplan-Meier method.Incidence of Core mutation I97L differed significantly among groups: A, 30% (3/10); B, 36.4% (4/11); C, 83.3% (10/12) (p = 0.021). Cumulative incidences of persistent low HBV DNA and HBsAg clearance were significantly higher in patients with I97L than in those with wild-type I97 (p = 0.003 and p = 0.016, respectively), and even higher in those with P79Q.In patients with CHB, measurement of I97L and additional mutation P79Q would be useful for predicting persistent low HBV DNA, normal ALT, and HBsAg clearance.

To investigate the lymphomagenesis of NK/T lymphoma, we comprehensively and systematically analyzed the expression pattern of the human NK/T cell line (NK-YS) genome by cDNA expression array and tissue microarray. We detected significant changes in the gene expression of NK-YS cell line: an increase in 18 and a decrease in 20 genes compared to normal NK cells or peripheral blood mononuclear cells. Among these genes, we found a strong decrease in hematopoietic cell specific protein-tyrosine-phosphatase SH-PTP1 (SHP1) mRNA by cDNA expression array and reverse transcriptase-polymerase chain reaction. Further analysis with standard immunohistochemistry and tissue microarray, which used 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphoma specimens and more than 95% of various types of malignant lymphoma were negative for SHP1 protein expression. On the other hand, SHP1 protein was strongly expressed in the mantle zone and interfollicular zone lymphocytes in reactive lymphoid hyperplasia specimens. In addition, various kinds of hematopoietic cell lines, particularly the highly aggressive lymphoma/leukemia lines, lacked SHP1 expression in vitro, suggesting that loss of SHP1 expression may be related to not only malignant transformation, but also tumor cell aggressiveness. SHP1 expression could not be induced in either of two NK/T cell lines by phorbol ester, suggesting that genetic impairment or modification with methylation of SHP1 DNA could be one of the critical events in the pathogenesis of NK/T lymphoma. This evidence strongly suggests that loss of SHP1 gene expression plays an important role in multistep tumorigenesis, possibly as an anti-oncogene in the wide range of lymphomas/leukemias as well as NK/T lymphomas.

Allogeneic leukocyte immunotherapy is often used to improve fertility of patients with habitual abortion (HA), which probably acts through immune-mediated mechanisms. However, the involvement of T cells is not clear. This study examined the effect of allogeneic immunotherapy on T cells of patients with HA. Peripheral blood mononuclear cells were obtained from 5 healthy women and 14 women with HA. RNA was isolated from mononuclear blood cells. Reverse transcription-polymerase chain reaction (RT-PCR), followed by single-strand conformational polymorphism (SSCP) were used to analyze the gene segments of T-cell receptor β chain (TCRβV) variable regions. Oligoclonal accumulation of T cells was identified in peripheral blood of nonpregnant patients with a history of HA. It was also revealed, however, that immunostimulation reduced the number of accumulating T-cell clones (p = 0.0004). The results, together with the clinical effectiveness of immunotherapy, suggest that accumulation of T-cell clonotypes, which probably resulted from antigenic stimulation, is involved in the pathogenesis of HA.

Epstein-Barr virus-associated hemophagocytic syndrome (EBV-AHS) is often associated with fatal infectious mononucleosis. However, the animal model for EBV-AHS has not been developed. We reported the first animal model for EBV-AHS using rabbits infected with EBV-related herpesvirus of baboon (HVP). Eleven of 13 (85%) rabbits inoculated intravenously with HVP-producing cells developed fatal lymphoproliferative disorders (LPD) between 22 and 105 days after inoculation. LPD was also accompanied by hemophagocytic syndrome (HPS) in nine of these 11 rabbits. The peroral spray of cell-free HVP induced the virus infection with increased anti-EBV-viral capsid antigen-IgG titers in three of five rabbits, and two of these three infected rabbits died of LPD with HPS. Autopsy revealed hepatosplenomegaly and swollen lymph nodes. Atypical lymphoid T cells expressing EBV-encoded small RNA-1 infiltrated diffusely in many organs, frequently involving the lymph nodes, spleen, and liver. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. HVP-DNA was detected in the tissues and peripheral blood from the infected rabbits by polymerase chain reaction or Southern blot analysis. Reverse transcriptase-polymerase chain reaction revealed both HVP-EBNA1 and HVP-EBNA2 transcripts, suggesting latency type III infection. These data indicate that the high rate of rabbit LPD with HPS induction is caused by HVP. This system is useful for studying the pathogenesis, prevention, and treatment of human EBV-AHS.