Search

Hepatic progenitor cells, called hepatoblasts, are highly proliferative and exhibit bipotential differentiation into hepatocytes and cholangiocytes in the fetal liver. Thus, they are the ideal source for transplantation therapy. Although several studies have been performed in vitro, the molecular mechanisms regulating hepatoblast differentiation in vivo following transplantation remain poorly understood. The aim of this study was to investigate an in vivo model to analyze hepatoblast bipotency and proliferative ability.Hepatic transplantation model using Cre-inducible diphtheria toxin receptor-transgenic mice (iDTR), and albafpCre mice expressing Cre under the control of albumin and α-fetoprotein (AFP) regulatory elements were established. Fresh hepatoblasts were transplanted into diphtheria toxin (DT)-injected iDTRalbafpCre mice and we analyzed their differentiation and proliferation abilities by immunostaining and gene expression profiles.Fresh hepatoblasts transplanted into DT-injected iDTRalbafpCre mice engrafted and differentiated into both hepatocytes and cholangiocytes. Additionally, the number of engrafted hepatoblast-derived hepatocytes increased following partial hepatectomy and serial DT injections. Expression levels of hepatic functional genes in transplanted hepatoblast-derived hepatocytes were similar to that of normal hepatocytes.In our iDTRalbafpCre transplantation model, fresh hepatoblasts could differentiate into hepatocytes and cholangiocytes. In addition, these donor cells were induced to proliferate by the following liver injury stimulation. This result suggests that this model is valuable for investigating hepatoblast differentiation pathways in vivo.

Hepatoblasts are somatic progenitor cells in the fetal liver, which retain a high proliferative capacity and differentiate into both hepatocytes and cholangiocytes in vivo. Although efficient expansion of hepatoblasts in vitro has been difficult without genetic modification, we have previously demonstrated that the interaction with mesenchymal cells is important for expansion of hepatoblasts in vitro. In this study, we show cell signaling pathways regulating the long-term proliferative ability of hepatoblasts. Individual primary hepatoblasts derived from mouse fetal livers formed large colonies when cocultured with mesenchymal feeder cells; however, secondary colony formation was unsuccessful, indicating that in vitro culture could induce short-term, but not long-term, proliferation. When the MEK inhibitor, PD0325901, was added to these cultures, hepatoblasts formed large colonies containing many Ki-67-positive cells. Expression of p16/19(cdkn2a), a cyclin-dependent kinase inhibitor, was induced after 3-6 days culture of hepatoblasts, whereas PD0325901 significantly suppressed this expression. Consistent with these observations, fetal hepatoblasts derived from p16/19(cdkn2a) knockout mice showed long-term proliferation without PD0325901, suggesting that MEK activity induced cell cycle arrest through accumulation of p16/19(cdkn2a). In transplantation assays, we could demonstrate that in vitro expanded hepatoblasts could proliferate and differentiate into hepatocytic and cholangiocytic cells in injured livers. It should also be noted that ERK in primary hepatoblasts was not highly activated during fetal liver development. Collectively, all these findings suggest that the MEK/ERK-independent pathway in the fetal liver is involved in hepatoblast proliferation to avoid accumulation of cyclin-dependent kinase inhibitor.

Background and Purpose— We investigated the factors influencing inpatient convalescent rehabilitation outcomes in patients with ischemic stroke, particularly severity of leukoaraiosis on magnetic resonance imaging. Methods— Participants included 520 patients with ischemic stroke (317 men and 203 women; mean age, 72.8±8.4 years) who were transferred from acute care hospitals for inpatient convalescent rehabilitation. Ischemic stroke subtypes included lacunar infarction (n=41), atherothrombosis (n=223), artery-to-artery embolism (n=67), cardiogenic embolism (n=97), undetermined embolism (n=76), and uncategorized ischemic stroke (n=16). Leukoaraiosis was graded according to periventricular hyperintensity (PVH) and deep white matter hyperintensity on magnetic resonance imaging. Functional Independence Measure scores were assessed on admission and at discharge. Results— Multiple regression analysis revealed that rehabilitation outcomes, measured as total Functional Independence Measure scores, were significantly associated with leukoaraiosis estimated by PVH grade. This association was observed after adjustment for factors such as severity, age, and poststroke history. In all patients, PVH grades were associated with Functional Independence Measure motor scores ( P P Conclusions— Our study revealed that the degree of leukoaraiosis was associated with inpatient convalescent rehabilitation outcome in patients with ischemic stroke. Furthermore, the PVH grade was associated with motor function outcome, whereas the deep white matter hyperintensity grade correlated with cognitive function outcome, likely because the progression patterns and anatomic backgrounds of PVH and deep white matter hyperintensity differ according to ischemic stroke subtype.

Objective Sodium-glucose cotransporter 1 (SGLT1) is thought to be expressed in the heart as the dominant isoform of cardiac SGLT, although more information is required to delineate the subtypes of SGLTs in human hearts. Moreover, the functional role of SGLTs in the heart remains to be fully elucidated. We herein investigated whether SGLT1 is expressed in human hearts and whether SGLTs significantly contribute to cardiac energy metabolism during ischemia-reperfusion injury (IRI) via enhanced glucose utilization in mice. Methods and Results We determined that SGLT1 was highly expressed in both human autopsied hearts and murine perfused hearts, as assessed by immunostaining and immunoblotting with membrane fractionation. To test the functional significance of the substantial expression of SGLTs in the heart, we studied the effects of a non-selective SGLT inhibitor, phlorizin, on the baseline cardiac function and its response to ischemia-reperfusion using the murine Langendorff model. Although phlorizin perfusion did not affect baseline cardiac function, its administration during IRI significantly impaired the recovery in left ventricular contractions and rate pressure product, associated with an increased infarct size, as demonstrated by triphenyltetrazolium chloride staining and creatine phosphokinase activity released into the perfusate. The onset of ischemic contracture, which indicates the initiation of ATP depletion in myocardium, was earlier with phlorizin. Consistent with this finding, there was a significant decrease in the tissue ATP content associated with reductions in glucose uptake, as well as lactate output (indicating glycolytic flux), during ischemia-reperfusion in the phlorizin-perfused hearts. Conclusions Cardiac SGLTs, possibly SGLT1 in particular, appear to provide an important protective mechanism against IRI by replenishing ATP stores in ischemic cardiac tissues via enhancing availability of glucose. The present findings provide new insight into the significant role of SGLTs in optimizing cardiac energy metabolism, at least during the acute phase of IRI.

Objective : The purpose of this study was to examine the effect of sleep-wake rhythm on improvements in level of consciousness disturbance for patients with cerebrovascular disease who received sensory stimulation care. Methods : The sample comprised four patients (3 men, 1 woman) with cerebrovascular disease who were hospitalized. Using sleep-stage analysis by means of 24-hour continuous electroencephalography by polysomnogram, the relationship between the establishment of sleep-wake rhythm and change in level of consciousness disturbance was examined. Results : Some measurements indicated that the establishment of the sleep-wake rhythm may be effective for improving the level of consciousness of patients who are receiving the sensory stimulation. However, it was difficult to verify this hypothesis because it was impossible to control the effects of other variables considered to contribute to the regulation of consciousness Conclusions : These findings provide a useful basis for future research using a more precise method.

Aim: C-reactive protein (CRP) elevation is reportedly a prognostic factor in patients with renal cell carcinoma (RCC). Thrombocytosis has recently been reported also to be a prognostic factor in RCC and, like CRP, to be related to inflammatory cytokines such as interleukin-6. The aim of this study was to evaluate the importance of both thrombocytosis and CRP elevation in tumor recurrence and prognosis for patients with RCC. Methods: The clinical records of 178 patients who underwent radical nephrectomy were reviewed. Thrombocytosis was defined as a platelet count ≥350 000/mm3, and CRP elevation was defined as a CRP level ≥1.0 mg/dL. Disease-free survival and cause-specific survival rates were calculated. Independent predictors for recurrence and prognosis were determined. Results: Patients with thrombocytosis and patients with elevated CRP levels had significantly higher pathological T stage, clinical stage, tumor size, histological grade, and percentage of microvascular invasion than did patients without THC and patients with CRP levels

Full-length sequences were determined for a human hepatitis E virus (HEV) isolate (HE-JA04-1911) and two swine HEV isolates (swJ8-5 and swJ12-4) that belong to one of three clusters within genotype 3 in Japan and are close to Spanish isolates according to their partial sequences. The three HEV isolates were 89.7–92.9 % identical to each other, but only 80.7–83.0 % similar to 21 HEV strains of the same genotype isolated in Canada, Kyrgyzstan, the USA and Japan over their entire genome. On comparison with HEV isolates whose partial sequence is known, the HE-JA04-1911, swJ8-5 and swJ12-4 isolates segregated into a phylogenetic cluster consisting of human and swine HEV isolates in Japan and the UK, with identities of 89.8–100 % and 87.9–92.4 %, respectively. Genotype 3 HEV isolates were found to be markedly heterogeneous. The UK-isolate-like HEV strains in Japan may have originated from the UK via the importation of pigs since 1900.

The ubiquitin-proteasome pathway is responsible for regulating cell cycle proteins, tumor-suppressor molecules, oncogenes, transcription factors, and pro- and anti-apoptotic proteins. The aim of this study is to evaluate the effects of proteasome inhibitors on human hepatocellular carcinoma (HCC) cells. HCC cells SK-Hep1, HLE and HepG2 were treated with the proteasome inhibitors MG132 and MG115. Our data showed that both inhibitors induce apoptosis in the three cell types tested in a dose-dependent manner. Moreover, subtoxic levels of MG132 and MG115 sensitized HCC cells to TRAIL-induced apoptosis. To investigate the mechanism of increased TRAIL sensitivity in HCC cells, we first examined surface expression of TRAIL and its receptors. MG132 upregulated TRAIL and its receptors (TRAIL-R1 and -R2) in SK-Hep1 and HLE, whereas MG115 upregulated them in SK-Hep1. MG132 downregulated expression of X-linked inhibitor of apoptosis protein (XIAP) in SK-Hep1 and HLE, and of survivin in all three cell-types. MG115 downregulated expression of XIAP in SK-Hep1, and survivin in SK-Hep1 and HepG2. Furthermore, MG132 downregulated phospho-AKT and its downstream target phospho-BAD, indicating that MG132 activated the mitochondrial apoptosis pathway by inhibiting phosphorylation of AKT and BAD. In conclusion, proteasome inhibitors induced apoptosis and augmented TRAIL sensitivity via both the IAP family and AKT pathways. Thus, combining proteasome inhibitors with a TRAIL agonist may provide a new therapeutic strategy for HCC.

28歳の男性. 2003年5月, 以前から指摘されていた左内シャント動脈瘤をネコに咬まれ受傷した. 左前腕部の腫脹, 熱感, 疼痛のため当科を受診した. 来院時, 動脈瘤の直上に2箇所の咬傷を認め, 内シャントは完全に閉塞していた. 蜂窩織炎の疑いで緊急入院となった. amikacin, piperacillin, clindamycinを併用し, γ-グロブリンを投与した. 後日, 創培養からパスツレラが検出されたためpanipenem/betamipronとminocyclineの併用に変更した. 炎症反応は改善傾向にあったが, 22病日に動脈瘤が突然破裂した. 用手的に圧迫止血し, 緊急に内シャント動脈瘤切除を施行した. 28病日に左肘部に内シャントを再建し, 維持透析を行っている. ネコ咬傷後のパスツレラ感染症は増加しているが, シャント感染をきたした報告例は世界初と思われる.

肝硬変に伴う脾機能亢進症による汎血球減少症の改善および門脈圧減圧を目的に施行した脾摘術後に, 汎血球減少症のみならず肝予備能の改善を認め, 肝不全状態を離脱できたアルコール性肝硬変例およびB型肝硬変例 (HBsAg seroconversion後) を経験した. 現在のところ非代償性肝硬変例において肝予備能を改善する唯一の治療法は肝移植であるが, 我々の経験から脾機能亢進症を伴う非代償性肝硬変で生体肝移植のドナーが確保できない場合, 肝予備能改善目的の代替治療の一つとして脾摘術を考慮してよいと考えられた. しかし, 癌およびウイルスに対する影響については未確認であるため肝癌合併例または肝炎ウイルス存在例では慎重に適応を検討する必要があると考えられる.

We report a case of primary scrotal sclerosing lipogranuloma. A 39-year-old man who had complained of a painless intrascrotal mass was introduced to our hospital for detailed examinations. He denied having received any injection of exogenous substances or having suffered from any trauma. Physical examination revealed a U-shaped elastic hard mass surrounding the penile shaft in the scrotum. T2-weighted magnetic resonance imaging (MRI) demonstrated an ill-defined U-shaped lesion which exhibited relatively low signal intensity. Primary scrotal sclerosing lipogranuloma was the most suspected. The mass gradually disappeared after 47 days from tumor open biopsy for definitive diagnosis. We found 227 cases reported in Japan, and we discuss the diagnosis, treatment and clinical features with reference to previous reports.

Summary Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells, but not in most normal cells. The role of TRAIL in hepatic cell death and hepatic diseases is not well understood. The present study investigated the expression of TRAIL and TRAIL receptors (TRAIL-Rs) in patients with hepatitis C virus infection using immunohistochemistry and examined physiological roles under viral infection in the HepG2 cell line. Staining of TRAIL or TRAIL-Rs was prominent in the cytoplasm and membrane of hepatocytes in the periportal area. Some liver-infiltrating lymphocytes also displayed positive staining for TRAIL. Staining intensity was significantly increased with disease progression, particularly in the periportal area. AdCMVLacZ (Q-BIOgene, Carisbad, Calif) infection was also found to induce apoptosis in HepG2 cells and significantly augment TRAIL-induced apoptosis. Anti-TRAIL antibody significantly inhibited apoptosis induced by AdCMVLacZ infection. Flow cytometry analysis revealed that both TRAIL-R2 and TRAIL were up-regulated on the cell surface of HepG2 cells with AdCMVLacZ infection. Transforming growth factor– β 1 also enhanced TRAIL expression in HepG2 cells. These results indicate that TRAIL/TRAIL-R apoptotic pathways play important roles in the hepatic cell death during viral infection.

Dietary arginine supplementation attenuates renal damage after relief of unilateral ureteral obstruction in rats. Background Progression of renal injury after relief of unilateral ureteral obstruction (UUO) has been demonstrated. Nitric oxide (NO) may be an effective intervention due to its vasodilatory, antifibrotic, and anti-apoptotic effects. Herein, we used dietary L-arginine (ARG) supplementation in a UUO relief model. Methods This study comprised group 1, control (no treatment). All other rats were subject to 3-day UUO, which was then relieved, and the rats maintained for 7 additional days. Group 2, no additional treatment; group 3, L-ARG; group 4, L-NAME, NO synthase inhibitor; group 5, ARG and L-NAME. Urinary NO 2/3 was quantified. GFR and ERPF were measured at day 10. Interstitial fibrosis and fibroblast expression, macrophage infiltration, tubular apoptosis, and proliferation, NOS expression, and the levels of tissue TGF-β were evaluated. Results Urinary NO 2/3 was significantly increased by ARG treatment and decreased by L-NAME. GFR and ERPF measured 7 days following relief were not significantly different in the previously obstructed kidneys (POK) of groups 2 and 3. L-NAME significantly reduced GFR and ERPF in the POK. ARG significantly reduced apoptosis, macrophage infiltration, and fibroblast expression in the POK. L-NAME exacerbated the effects on apoptosis and fibroblasts. Fibrosis was minimal in groups 1 through 3, but was significantly increased by L-NAME. ARG did not affect renal NOS expression and tissue TGF-β1 levels. Conclusion Dietary ARG supplementation during UUO relief did not improve ERPF or GFR. However, renal damage, including fibrosis, apoptosis, and macrophage infiltration was significantly improved by ARG treatment. This suggests that increasing NO availability could be beneficial in the setting of UUO relief.

INTRODUCTION Waterston et al. have classified the risk of morbidity in infants with esophageal atresia and tracheoesophageal fistula. However, few cases of esophageal atresia with distal tracheoesophageal fistula in extremely low birth weights infants have been reported. In such infants, the selection of primary reconstruction or staged repair remains controversial. In the present report, we describe the difficulties of perioperative management of such small infants and discuss how to rescue them. PRESENTATION OF CASE A 471-g female infant was delivered at 28 weeks’ gestation via cesarean section. Esophageal atresia with distal tracheoesophageal fistula was diagnosed. Esophageal banding and gastrostomy were performed via laparotomy on day 1. On day 74, when the infant weighed almost 1000 g, airway management was discontinued. On day 136, endotracheal intubation again became necessary because of respiratory problems, and the esophagus was reconstructed on day 141. Despite this operation, the patient died on day 276 because of continuing respiratory problems. DISCUSSION Esophageal banding is considered an appropriate treatment for respiratory problems in such extremely low weight infants. However, the timing of dissection of the tracheoesophageal fistula after esophageal banding is extremely important. CONCLUSION In the present case, ligation of the tracheoesophageal fistula and esophageal reconstruction should have been performed as soon as possible.

Progression of renal damage after relief of unilateral ureteral obstruction (UUO) has been demonstrated, especially in neonatal rats. We evaluated renal function and renal damage after relief of 3-day UUO in five groups of adult rats: group 1, no treatment; group 2, 3-day UUO; groups 3- 5, 3-day UUO followed by relief; group 3, 7-day relief; group 4, 14-day relief; and group 5, 28-day relief. Glomerular filtration rate (GFR), renal blood flow (RBF), tissue transforming growth factor-β (TGF-β), interstitial fibrosis and fibroblast expression, tubular apoptosis, macrophage infiltration, expression of nitric oxide synthases (NOS), and urinary nitrate/nitrite (NO2/NO3) were evaluated. RBF and GFR were decreased to 1and interstitial fibrosis were significantly higher in POK of groups 3- 5 compared with groups 1 and 2 . In group 5, the numbers of infiltrating macrophages, fibroblasts, and apoptotic tubular cells were higher in POK compared with group 1. Urinary NO2/NO3was significantly higher than baseline from 3 to 27 days after relief of UUO. Expression of NOS isoforms was increased in tubules. As interstitial fibrosis contributes to decreased renal function, these results suggest that the acute recovery in function may be compromised in the long term by the progressive renal fibrosis which was found. Furthermore, pharmacological intervention at the time of relief of UUO, targeted to fibrotic processes, may contribute to long-term recovery of renal function.

Liposome-mediated transfer of nitric oxide synthase gene improves renal function in ureteral obstruction in rats Background The protective effect of nitric oxide has been demonstrated in several renal disease models. We augmented renal nitric oxide production by transfer of the inducible nitric oxide synthase (iNOS) gene into rat kidney in controls and in unilateral ureteral obstruction (UUO). Methods The human iNOS gene was inserted into a pcDNA 3.1-backbone plasmid with the FLAG epitope (FLAG-iNOS). In vitro, transduction of FLAG-iNOS was confirmed by Western blot and Griess reaction. In vivo, we transfected either FLAG-iNOS or control plasmid (CMV-LacZ), using cationic liposomes. Urinary nitric oxide metabolites and immunohistochemistry confirmed iNOS transduction. Renal function was also assessed. Results In vitro, increased iNOS expression was demonstrated in human embryonic kidney (HEK293) cells, along with increased release of nitric oxide metabolites, NO2/NO3. In vivo, FLAG-iNOS was detected by polymerase chain reaction (PCR) up to 35 days after the transfection. Urine collection documented increased urinary NO2/NO3. Immunohistochemistry localized iNOS to collecting ducts, distal tubules, and glomerulus of the injected kidney. Renal function measured up to 21 days after transfection in control animals was not significantly different between the two groups. In contrast, renal function after 24 hours of UUO was significantly improved in FLAG-iNOS–treated animals. Conclusion This study demonstrates the feasibility of liposome-mediated iNOS gene transfer into the kidney. Furthermore, the improvement of renal function in UUO demonstrates that the transfected iNOS gene is active and suggests that decreased iNOS activity contributes to the decreased renal function in UUO. This iNOS construct may have therapeutic utility in the pathophysiologic sequelae of UUO and other renal diseases.

We report a female case of septic endophthalmitis and meningitis associated with Klebsiella pneumoniae liver abscess which was thought to be caused by duodeno-biliary reflux related to choledochoduodenostomy. We treated this patient by ultrasonography-guided percutaneous abscess drainage and intravenous administration of third generation antibiotics. However, the visual function of her left eye was eventually lost. Reports of liver abscess with metastatic lesions are rare, and our experience suggests that more physicians should be alert to septic metastatic lesions such as K. pneumoniae liver abscess or bacteremia with complaints of ocular or central nervous system symptoms.

Purpose The International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) risk model has been designed for prognostification in patients with metastatic renal cell carcinoma (mRCC) treated with targeted therapy. One factor is neutrophil count; however, increasing evidence has suggested the superiority of neutrophil-to-lymphocyte ratio (NLR) for predicting outcome. In this study, we evaluate the prognostic effect of NLR levels on patients with mRCC treated with targeted therapy, and then we compare the predictive accuracy of the IMDC risk model and its modified one by using NLR, instead of neutrophil count. Patients and method A total of 277 patients are included for the analysis. All patients underwent targeted therapies and associated outcome are assessed using multivariate analysis. Results Pretreatment NLR levels are elevated in 30.3% and 23.1% of patients in the first-line and subsequent second-line setting, respectively. Kaplan-Meier curves reveal that elevated pretreatment NLR is significantly associated with poor overall survival (OS) since first-line (P Conclusion Changes in NLR levels could be predictive for prognosis in patients with mRCC treated with first-line and second-line targeted therapy. The addition of NLR significantly improves the predictive accuracy of the IMDC risk model in the first-line and subsequent second-line setting.

Fetal hepatic stem/progenitor cells, hepatoblasts, are highly proliferative cells and the source of both hepatocytes and cholangiocytes. In contrast, mature hepatocytes have a low proliferative potency and high metabolic functions. Cell proliferation is regulated by cell cycle-related molecules. However, the correlation between cell cycle regulation and hepatic maturation are still unknown. To address this issue, we revealed that the cell cycle inhibitor p57(Kip2) was expressed in the hepatoblasts and mesenchymal cells of fetal liver in a spatiotemporal manner. In addition, we found that hepatoblasts in p57(Kip2)-/- mice were highly proliferative and had deficient maturation compared with those in wild-type (WT) mice. However, there were no remarkable differences in the expression levels of cell cycle- and bipotency-related genes except for Ccnd2. Furthermore, p57(Kip2)-/- hepatoblasts could differentiate into mature hepatocytes in p57(Kip2)-/- and WT chimeric mice, suggesting that the intrinsic activity of p57(Kip2) does not simply regulate hepatoblast maturation.

The prognosis of patients with renal cell carcinoma (RCC) and liver metastasis (LM) is poor. We evaluated the clinical characteristics, prognosis and prognostic factors of RCC patients with LM. A total of 25 patients who underwent radical or partial nephrectomy (Nx) for RCC between November, 1980 and April, 2013 at the National Defense Medical College, Tokorozawa, Saitama, Japan, with LM at initial presentation or following Nx, were included in this study. The association between prognosis following development of LM and clinicopathological parameters was analyzed. The Cox proportional hazards regression model was used to identify prognostic predictors. The median cancer-specific survival (CSS) following LM diagnosis was 10.6 months. The presence of sarcomatoid differentiation, Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2, C-reactive protein ≥1.0 mg/dl, corrected calcium ≥10 mg/dl and presence of multiple organ metastases, were identified as CSS predictors. The multivariate analysis identified ECOG PS ≥2 as an independent CSS predictor. Nine patients survived for >20 months following LM diagnosis and 1 patient, who received treatment with tyrosine kinase inhibitors (TKIs) for LM, exhibited stable disease for 5 years. Nine patients underwent local LM treatment. Two patients, who underwent hepatic resection, survived for 55.1 and 22 months, respectively. In conclusion, RCC patients with LM may benefit from local LM treatment if they have a limited number of metastases in addition to LM and if their ECOG PS is satisfactory. Indeed, a proportion of RCC patients with LM benefit from TKI therapy. Furthermore, RCC patients with LM and ECOG PS ≥2 apparently have a poor prognosis, regardless of local or systemic therapies.

Background Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. Inhibition of histone deacetylase (HDAC) 6 by the HDAC inhibitor panobinostat hinders the refolding of unfolded proteins by increasing the acetylation of heat shock protein 90. We investigated whether combining panobinostat with the proteasome inhibitor bortezomib would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins, thereby causing ubiquitinated proteins to accumulate and induce ER stress. Methods Caki-1, ACHN, and 769-P cells were treated with panobinostat and/or bortezomib. Cell viability, clonogenicity, and induction of apoptosis were evaluated. The in vivo efficacy of the combination was evaluated using a murine subcutaneous xenograft model. The combination-induced ER stress and ubiquitinated protein accumulation were assessed. Results The combination of panobinostat and bortezomib induced apoptosis and inhibited renal cancer growth synergistically (combination indexes

Identification of genes specifically expressed in stem/progenitor cells is an important issue in developmental and stem cell biology. Genome-wide gene expression analyses in liver cells performed in this study have revealed a strong expression of X-linked genes that include members of the brain-expressed X-linked (Bex) gene family in stem/progenitor cells. Bex family genes are expressed abundantly in the neural cells and have been suggested to play important roles in the development of nervous tissues. However, the physiological role of its individual members and the precise expression pattern outside the nervous system remain largely unknown. Here, we focused on Bex2 and examined its role and expression pattern by generating knock-in mice; the enhanced green fluorescence protein (EGFP) was inserted into the Bex2 locus. Bex2-deficient mice were viable and fertile under laboratory growth conditions showing no obvious phenotypic abnormalities. Through an immunohistochemical analysis and flow cytometry-based approach, we observed unique EGFP reporter expression patterns in endocrine and stem/progenitor cells of the liver, pyloric stomach, and hematopoietic system. Although Bex2 seems to play redundant roles in vivo, these results suggest the significance and potential applications of Bex2 in studies of endocrine and stem/progenitor cells.

Warfarin is widely used to perform catheter ablation for atrial fibrillation (AF). Heparin is usually administered during this procedure to prevent thromboembolic events, while protamine is used to reduce the incidence of bleeding complications. The purpose of this study was to investigate the influence of heparin and protamine administration on the effects of warfarin and its safety. The subjects included 226 AF patients (206 males, 54.9 ± 9.1 years, paroxysmal/persistent AF: 118/108) undergoing AF ablation with the discontinuation of warfarin administration over 2 days. Heparin was administered to achieve an activated clotting time (ACT) above 300 s during the procedure. Several parameters of the coagulation status, including the prothrombin time international normalized ratio (PT-INR) and ACT values, measured immediately before and after protamine infusion were compared. The mean value of PT-INR prior to ablation was 1.9 ± 0.6. At the end of the procedure, the mean ACT and PT-INR values were 348.0 ± 52.9 and 2.9 ± 0.7, respectively. Following the infusion of 30 mg of protamine, both the ACT and PT-INR values significantly decreased, to 159.6 ± 31.0 (p < 0.0001) and 1.6 ± 0.3 (p < 0.0001), respectively. No cases of symptomatic cerebral infarction were observed, although femoral hematomas developed in 17 (7.5 %) of the patients without further consequence. The concomitant use of heparin augments the effect of warfarin. Meanwhile, protamine administration immediately reverses both the ACT and PT-INR, indicating the applicability of protamine for AF ablation in patients under the mixed administration of heparin and warfarin.

Although catheter ablation targeting the pulmonary vein (PV) is a well-known therapy for patients with paroxysmal atrial fibrillation (PAF), ectopic firings from the superior vena cava (SVC) can initiate PAF. The purpose of this study was to investigate predictors of SVC firing. The subjects included 336 consecutive PAF patients (278 males, age 56.1 ± 10.8 years) undergoing atrial fibrillation (AF) ablation. The appearance of SVC firing was monitored throughout the procedure using a decapolar catheter with multiple electrodes to record electrograms of the coronary sinus and SVC. In addition to PV isolation, SVC isolation was performed only in patients with documented SVC firing. SVC firing was observed in 43/336 (12.8 %) of the patients, among whom complete isolation of the SVC was achieved in 40/43 (93 %) patients. A lower body mass index (BMI) (22.8 ± 2.8 vs 24.1 ± 3.1 kg/m2, p = 0.007) and higher prevalence of prior ablation procedures (58 vs 18 %, p = 0.0001) were related to the presence of SVC firing. In a multivariate analysis, a lower BMI (p = 0.012; odds ratio 0.83, 95 % CI 0.72 to 0.96) and history of prior ablation procedures (p

There is currently no curative treatment for advanced renal cancer. Enhancing histone acetylation is a promising epigenetic-based therapy for cancer; however, in solid tumors, the efficacy of histone deacetylase (HDAC) inhibitors alone is limited. The human immunodeficiency virus protease inhibitor ritonavir is also a CYP3A4 inhibitor and we hypothesized that combining ritonavir with the HDAC inhibitor panobinostat, one of the substrates of CYP3A4, may effectively eliminate renal cancer cells by enhancing the activity of panobinostat. The combination of ritonavir and panobinostat synergistically inhibited cancer cell growth and cancer cell colony formation, while only slightly inhibiting the growth of renal proximal tubule epithelial cells. This combination significantly induced apoptosis in cancer cells and this apoptosis was considered to be caspase-dependent, since the pan-caspase inhibitor Z-VAD-FMK reduced the number of Annexin V-positive cells. In murine subcutaneous xenograft models using Caki-1 cells, a 10-day treatment with the combination of ritonavir and panobinostat significantly inhibited tumor growth. Panobinostat alone increased histone acetylation in a dose-dependent manner and the co-administration of ritonavir synergistically enhanced this acetylation. Furthermore, this combination inhibited the expression of HDACs, which may also play a role in the enhancement of histone acetylation. Thus, the present study may provide a basis for testing the combination of ritonavir and panobinostat for patients with advanced renal cancer.

INTRODUCTION AND OBJECTIVES: Ritonavir inhibits proteasomes and P-glycoprotein, an efflux pump associated with multidrug resistance in renal cancer. The histone deacetylase (HDAC) inhibitor romidepsin increases the amount of unfolded proteins in cells by suppressing the function of molecular chaperones. We thought that ritonavir and romidepsin together would induce histone acetylation and endoplasmic reticulum (ER) stress synergistically because ritonavir would enhance romidepsin’s activity by inhibiting its efflux from cells and would also enhance the accumulation of romidepsin-increased unfolded proteins by inhibiting their degradation. METHODS: After renal cancer cells (ACHN, Caki-1, 769-P, 786-O) were treated with ritonavir (25-50 mM) and/or romidepsin (10-20 nM), cell viability and clonogenicity were assessed by MTS assay and colony formation assay. Induction of apoptosis and changes in the cell cycle were evaluated using flow cytometry. Histone acetylation, induction of ER stress, and the expression of HDACs and cell-cycle-associated proteins were evaluated by western blotting. RESULTS: Romidepsin was shown to be a P-glycoprotein substrate because the P-glycoprotein inhibitor PSC833 enhanced romidepsin-induced apoptosis, cytotoxicity, and histone acetylation but alone had no effect on apoptosis, cytotoxicity, or histone acetylation. Romidepsin combined with ritonavir exhibited strong cytotoxicity synergistically (combination indexes

combination synergistically caused ubiquitinated proteins to accumulate: 10 nM bortezomib alone induced no accumulation of ubiquitinated protein but in combination with 2 mM belinostat induced marked ubiquitinated protein accumulation, whereas belinostat alone did not change the amount of ubiquitinated proteins. The combination induced an unfolded protein response evidenced by the increased expression of glucose-regulated protein 78 and heat shock protein 70, and this confirmed the accumulation of ubiquitinated unfolded proteins in the cell. CONCLUSIONS: Bortezomib in combination with belinostat synergistically induces caspase-dependent apoptosis and inhibits renal cancer growth by causing ubiquitinated protein accumulation.

A total of 136 patients who underwent radical prostatectomy following histological diagnosis of prostate cancer by transrectal biopsy and 3-Tesla magnetic resonance imaging (MRI) were evaluated. MRI was performed on 26 patients before prostate biopsy (pre-biopsy group) and on 110 patients after prostate biopsy (post-biopsy group). We defined the largest tumor focus in a radical prostatectomy specimen as the index cancer. We compared the accuracy of MRI in detecting and localizing the index cancer in the groups. The sensitivity of detecting the index cancer by MRI was significantly (p = 0.012) higher in the pre-biopsy group (96.2%) than in the post-biopsy group (77. 3%). The negative predictive value of extracapsular invasion was 84.6% in the pre-biopsy group and 80.7% in the post-biopsy group. The average interval between biopsy and MRI was 42.8 days. Artifacts due to post-biopsy hemorrhage were observed in 32 (29.1%) of the patients in the post-biopsy group. The sensitivity of detecting the index cancer by MRI was significantly (p = 0.022) higher in 78 patients without artifacts due to hemorrhage (83.3%) than in the 32 patients with artifacts due to hemorrhage (62.5%). Even if MRI is delayed until after prostate biopsy,the artifact due to hemorrhage markedly interferes with the accuracy of MRI. Although pre-biopsy MRI is more accurate than post-biopsy MRI,there are some problems to be solved,such as cost effectiveness and the detectability of low-malignant and small cancers.

We determined whether the cyclooxygenase-2 inhibitor etodolac affects renal tubular damage and interstitial fibrosis in unilateral ureteral obstruction.Etodolac (10 mg./kg.) was administered to rats 1 day before unilateral ureteral obstruction and every day thereafter. Kidneys were harvested at day 14 after unilateral ureteral obstruction. Tissue transforming growth factor-beta and prostaglandin E2 were measured by bioassay using mink lung epithelial cells and enzyme linked immunosorbent-sandwich assay. Renal tubular proliferation and apoptosis were detected by immunostaining with proliferating cellular nuclear antigen and by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling, respectively. Cyclooxygenase-2 expression was detected by immunohistochemistry. Fibrosis was assessed by measuring collagen deposition in trichrome stained slides.Bioassay showed that in the control group obstructed kidneys contained significantly higher mean transforming growth factor-beta1 than unobstructed kidneys (79.1 +/- 8.3 versus 33.6 +/- 4.2 ng./gm. tissue) and etodolac significantly decrease the mean value in obstructed kidneys (46.2 +/- 10.0 ng./gm. tissue). Assay demonstrated that obstructed control kidneys had significantly more mean tubular apoptosis than their unobstructed counterparts (26.6 +/- 5.4 versus 2.2 +/- 1.4 nuclei per high power field) and etodolac significantly decreased mean renal tubular apoptosis in the obstructed kidneys (16.2 +/- 1.9 nuclei per high power field). In addition, immunostaining with proliferating cellular nuclear antigen showed that obstructed kidneys in the control group had significantly more mean renal tubular proliferation than unobstructed kidneys (9.8 +/- 3.4 versus 3.9 +/- 0.1 per high power field) and etodolac significantly increased mean proliferating renal tubule in the obstructed kidneys (24.9 +/- 4.3 per high power field). Control obstructed kidneys had significantly more fibrosis and prostaglandin E2 production, which were also significantly blunted by etodolac.The cyclooxygenase-2 inhibitor etodolac significantly reduces tissue transforming growth factor-beta, resulting in decreased tubular damage and interstitial fibrosis. This finding suggests that etodolac is a promising agent for preventing renal tissue damage in unilateral ureteral obstruction.

The influence of population density on salt preference was investigated in Sprague-Dawley rats. In experiment I, after 12 days of housing at one rat per cage, 12 female rats were divided randomly into two groups, i.e. a control group remaining at one rat per cage, and a crowded group housed at 3 rats per cage. The rats were observed for 21 days. Distilled water, and 0.5%, 1.0%, 1.5% saline solution were supplied ad libitum with salt-free feed. The salt intake of rats in the control groups did not change during the whole period. On the other hand, the salt intake of rats in the crowded group decreased significantly from 0.29±0.03 to 0.18±0.01g/rat/day (mean±SE, p

Some experiments for fundamental understanding of micro-phase separated structures formed in mixed monolayers of n-alkyltrichlorosilanes and a partially fluorinated amphiphile are presented. The surface morphology of the mixed monolayers is varied by changing some parameters, such as chain length of alkyltrichlorosilanes, temperature of the water surface, and solvents of spreading solutions. It is demonstrated that the surface structures are mainly governed by the dynamic molecular process during spreading. The most likely mechanism of the micro-phase separation is proposed.

The aim of this study was to assess the effect of plaunotol, an anti-ulcer agent, on trinitrobenzene sulfonic acid (TNB)- and acetic acid-induced colonic lesions in rats. Plaunotol significantly reduced the severity of colonic mucosal lesions induced by TNB at a dose of 600 mg/kg/day. Moreover, plaunotol, at a dose of 600 mg/kg/day, significantly depressed the myeloperoxidase activity of the lesioned area induced by TNB of the rat colon. In the model of colitis induced by acetic acid, plaunotol reduced the area of lesions dose-dependently and significantly at doses of 60, 200 and 600 mg/kg/day as assessed by macroscopic observation. Microscopic observation showed obvious changes by administration of plaunotol such as reduction of epithelial cell necrosis, decreased mucin production and a decreased infiltration of a large number of neutrophils. In conclusion, plaunotol showed a protective effect against colonic lesion formation induced by TNB and acetic acid in rats. This study suggests the possibility that plaunotol may be effective and useful for treatment of inflammatory bowel disease in humans.

This report is concerned with statistical analysis on long-term measurement of wave induced load of naval ships. We have analyzed wave-induced vertical bending stresses at midship deck and wave conditions measured by 12 naval ships which are equipped with Ship Motion Analyzing Computer System (SMACS) over a period of 84 years. We have introduced contribution rates between wave period and wave height on long term distribution of wave induced vertical bending stress in each stress revels and contribution rates between wave period and heading angle between ship and wave direction on the long term distribution. We derive the following conclusions.(1) When the range of the probability of exceedance in the long term distribution are from 10-1 to 10-2, the contribution rates between wave period and wave height and the rates between wave period and heading angle widely distribute over the wave conditions and heading angles.(2) For the range of the probability of exceedance from 10-4 to 10-8, the contribution rate is centered at a wave condition and a heading angle. The condition equals to the broad sense of resonance when the peaks of both the response amplitude function of the wave bending stress in regular wave and wave spectra are superimposed.

The Mitsunobu reaction of commercial 4-hydroxycoumarin with both (R)-(-)- and (S)-(+)-tert-butyl mandelates derived from commercial enantiopure mandelic acids furnished (S)-(+)- and (R)-(-)-tert-butyl O-coumarinylmandelates which were, then, treated with trifluoroacetic acid to give novel crystalline optically pure (S)-(+)- and (R)-(-)-O-coumarinylmandelic acids [SCMOH and RCMOH], respectively in good overall yields. Diastereotopic nonequivalence 1H-NMR examination of the resultant esters and amides without any racemization or kinetic resolution by way of a Steglich's procedure (a DCC-DMAP method) has proved each acid to be a useful, efficient and reliable chiral derivatizing agent for the enantiomeric excess determination of chiral alcohols and amines.

Objective Direct vision internal urethrotomy (DVIU) has been considered to be a low invasive and widely used therapeutic modality for male urethral stricture. However, its efficacy is still controversial. We herein evaluated the efficacy of DVIU for male urethral stricture. Patients and methods Nineteen patients 27 to 78 years old (median age = 59) underwent DVIU for urethral strictures at our hospital were included in this study. Strictures were at bulbar urethra in 17 patients, membranous urethra in 1 patient, and pendulous urethra in 1 patient. The stricture lengths estimated on retrograde urethrography were less than 1 cm in 13 patients, 1-2 cm in 2 patients, and more than 2 cm in 4 patients. The etiology of stricture was straddle injury in 7 patients, post transurethral surgery in 7 patients, pelvic fracture in 1 patient, and unknown in 4 patients. The operation was done by cold knife incision using guidewire. The duration of postoperative urethral catheterization was 5 to 35 days (mean 12.8 days). Follow up duration ranged from 1 month to 139 months (mean 48.2 months). The definition of postoperative re-stricture was the confirmation of re-stricture on retrograde urethrography or deterioration of symptom. Results While no severe complication was observed, postoperative re-stricture was seen in 13 patients. Stricture-free rates at 3 months, 6 months, and 5 years after the first DVIU were 44.4%, 38.1%, 20.3% respectively. Although second DVIU was done for 7 patients with re-stricture, six patients resulted in failure. Stricture-free rates at 3 months, 6 months, and 5 years after the second DVIU were 42.2%, 28.6%, 14.3% respectively. Though the third DVIU was done for two of them, they were unable to void just immediately after the removal of urethral catheters. Stricture-free rate in stricture less than 1 cm was higher than that in 1 cm or longer, though it did not reach significant difference (p = 0.1813). Conclusion The efficacy of DVIU is lesser than we expected. DVIU seems to be excessively applied to male urethral strictures and should not be performed for long and recurrent urethral stricture.

We examined whether fatty acid synthase (FAS) expression in prostate biopsy cores had valuable information and could predict a Gleason score (GS) upgraded from biopsy to radical prostatectomy (RP) specimens.Immunostaining with a FAS antibody was performed on paraffin-embedded prostate biopsy cores with GS 5-6 obtained from 80 patients who subsequently underwent RP. The correlations between FAS expression and clinicopathological parameters, upgrading group, and clinicopathological parameters including FAS expression were analyzed. Logistic regression analysis was performed to identify a significant set of independent predictors for upgrading GS.A total of 46 patients (57.5%) with biopsy GS 5-6 were upgraded to GS ≥7 at RP. FAS expression was significantly associated with clinical T stage (P = 0.0232) and positive core rate (P = 0.0245). Upgrading from biopsy GS 5-6 to GS ≥7 at RP was significantly associated with clinical T stage (P = 0.0337), positive core rate (P = 0.0262), and FAS expression (P 0.0001). FAS expression was a significant predictor for upgrading from biopsy GS 5-6 to GS ≥7 at RP in multivariate analysis (P 0.0001; odds ratio, 12.35). FAS scores showed the largest area under the receiver-operating characteristic curve (AUC) in preoperative parameters (AUC = 0.753).Increased FAS expression in prostate biopsy cores could be a novel parameter for upgrading from biopsy GS 5-6 to GS ≥7 at RP. If a biopsy GS is low, the treatment strategy for patients with high FAS expression in prostate biopsy cores should be carefully determined.

Background Intracellular sodium ([Na+]i) kinetics are involved in cardiac diseases including ischemia, heart failure, and hypertrophy. Because [Na+]i plays a crucial role in modulating the electrical and contractile activity in the heart, quantifying [Na+]i is of great interest. Using fluorescent microscopy with sodium-binding benzofuran isophthalate (SBFI) is the most commonly used method for measuring [Na+]i. However, one limitation associated with this technique is that the test cannot simultaneously evaluate the effects of several types or various concentrations of compounds on [Na+]i. Moreover, there are few reports on the long-term effects of compounds on [Na+]i in cultured cells, although rapid changes in [Na+]i during a period of seconds or several minutes have been widely discussed. Findings We established a novel technique for quantifying [Na+]i in cultured neonatal rat cardiomyocytes attached to a 96-well plate using a microplate reader in combination with SBFI and probenecid. We showed that probenecid is indispensable for the accurate measurement because it prevents dye leakage from the cells. We further confirmed the reliability of this system by quantifying the effects of ouabain, which is known to transiently alter [Na+]i. To illustrate the utility of the new method, we also examined the chronic effects of aldosterone on [Na+]i in cultured cardiomyocytes. Conclusions Our technique can rapidly measure [Na+]i with accuracy and sensitivity comparable to the traditional microscopy based method. The results demonstrated that this 96-well plate based measurement has merits, especially for screening test of compounds regulating [Na+]i, and is useful to elucidate the mechanisms and consequences of altered [Na+]i handling in cardiomyocytes.