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Purpose: Lower extremity magnetic resonance angiography (MRA) without electrocardiography (ECG) or peripheral pulse unit (PPU) triggering and contrast enhancement is beneficial for diagnosing peripheral arterial disease (PAD) while avoiding synchronization failure and nephrogenic systemic fibrosis. This study aimed to compare the diagnostic performance of turbo spin-echo-based enhanced acceleration-selective arterial spin labeling (eAccASL) (TSE-Acc) of the lower extremities with that of turbo field-echo-based eAccASL (TFE-Acc) and triggered angiography non-contrast enhanced (TRANCE)., Methods: Nine healthy volunteers and a patient with PAD were examined on a 3.0 Tesla magnetic resonance imaging (MRI) system. The artery-to-muscle signal intensity ratio (SIR) and contrast-to-noise ratio (CNR) were calculated. The arterial visibility (1: poor, 4: excellent) and artifact contamination (1: severe, 4: no) were independently assessed by two radiologists. Phase-contrast MRI and digital subtraction angiography were referenced in a patient with PAD. Friedman's test and a post-hoc test according to the Bonferroni-adjusted Wilcoxon signed-rank test were used for the SIR, CNR, and visual assessment. p < 0.05 was considered statistically significant., Results: No significant differences in nearly all the SIRs were observed among the three MRA methods. Higher CNRs were observed with TSE-Acc than those with TFE-Acc (anterior tibial artery, p = 0.014; peroneal artery, p = 0.029; and posterior tibial artery, p = 0.014) in distal arterial segments; however, no significant differences were observed upon comparison with TRANCE (all p > 0.05). The arterial visibility scores exhibited similar trends as the CNRs. The artifact contamination scores with TSE-Acc were significantly lower (but within an acceptable level) compared to those with TFE-Acc. In the patient with PAD, the sluggish peripheral arteries were better visualized using TSE-Acc than those using TFE-Acc, and the collateral and stenosis arteries were better visualized using TSE-Acc than those using TRANCE., Conclusion: Peripheral arterial visualization was better with TSE-Acc than that with TFE-Acc in lower extremity MRA without ECG or PPU triggering and contrast enhancement, which was comparable with TRANCE as the reference standard. Furthermore, TSE-Acc may propose satisfactory diagnostic performance for diagnosing PAD in patients with arrhythmia and chronic kidney disease., Competing Interests: Declaration of competing interest Makoto Obara is an employee of Philips Japan Ltd. The remaining authors have no other conflicts of interest related to this submission personally., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Parasitic helminths modify host immune reactions to promote long-term parasitism. We previously purified a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF), from the excretory/secretory products of Spirometra erinaceieuropaei plerocercoids and reported its cDNA and genomic DNA sequences. In this study, we isolated extracellular vesicles (EVs) from the excretory/secretory products of S. erinaceieuropaei plerocercoids and found that they suppressed the production of nitric oxide and the gene expression of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in lipopolysaccharide-stimulated macrophages. EVs are membrane-bound vesicles 50-250 nm in diameter and are localized in the whole bodies of plerocercoids. EVs from plerocercoids encapsulate a variety of unidentified proteins and microRNAs (miRNAs), which are non-coding RNAs that play essential roles in post-transcriptional gene regulation. The miRNAs of the EVs were analyzed, and 334,137 sequencing reads were mapped to the genomes of other organisms. A total of 26 different miRNA families were identified, including miR-71, miR-10-5p, miR-223, and let-7-5p, which have been reported to have immunosuppressive effects. We confirmed that P-ISF was present in the supernatant but not in the EVs by western blotting with an anti-P-ISF antibody. These results suggest that S. erinaceieuropaei plerocercoids suppress host immunity by releasing P-ISF and EVs., Competing Interests: Declaration of Competing Interest All authors have declared no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objective: Brain temperature monitoring using a catheter thermometer has been reported to be a useful technique to predict prognosis in neurosurgery. To investigate the possibility of measuring intracranial cerebrospinal fluid temperature for postoperative monitoring in patients with Moyamoya disease (MMD) after bypass surgery., Materials and Methods: This study evaluated fifteen patients with MMD who were indicated for bypass surgery. Diffusion tensor imaging for brain thermometry were performed on a 1.5-T MR scanner. Intracranial cerebrospinal fluid temperature with/without considering the fractional anisotropy component, body temperature, C-reactive protein levels, white blood cell count, and cerebral blood flow measured by 123 I-IMP single-photon emission computed tomography were obtained before surgery and 1-3 days after surgery. Pixel values considered to be signal outliers in fractional anisotropy processing were defined as cerebrospinal fluid noise index and calculated. Wilcoxon signed-rank test and effect size were performed to compare the changes before and after revascularization. Spearman's rho correlation coefficient was used to analyze the correlations between each parameter. Statistical significance was defined as p < 0.05., Results: All parameter values became significantly higher compared to those measured before revascularization (p < 0.01 in all cases). The effect sizes were largest for the cerebrospinal fluid temperature with fractional anisotropy processing and for C-reactive protein levels (Rank-biserial correlation = 1.0). The cerebrospinal fluid noise index and cerebrospinal fluid temperatures with fractional anisotropy processing (r = 0.84, p < 0.0001) or without fractional anisotropy processing (r = 0.95, p < 0.0001) showed highly significant positive correlations. Although no significant correlation was observed, cerebrospinal fluid temperatures with fractional anisotropy had small or moderately positive correlations with cerebral blood flow, body temperature, C-reactive protein levels, and white blood cell count (r = 0.37, 0.42, 0.41, and 0.44, respectively; p > 0.05)., Conclusion: Our findings suggest the possibility of postoperative monitoring for MMD patients by measuring intracranial cerebrospinal fluid temperature with fractional anisotropy processing. Intracranial cerebrospinal fluid temperature might be considered as combined response since cerebrospinal fluid, body temperature, and inflammation are equally correlated., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to compare the intraneural microvascular patterns of the ulnar nerve at 2 elbow flexion angles in asymptomatic volunteers and patients with cubital tunnel syndrome (CuTS) and to evaluate the effects of surgery on the microvascular pattern in patients with CuTS by using contrast-enhanced ultrasonography (CEUS)., Methods: This study included 10 elbows in 10 asymptomatic volunteers (control group) and 10 elbows in 10 patients with CuTS who underwent anterior subcutaneous transposition of the ulnar nerve (CuTS group). The CuTS group underwent clinical and electrophysiologic examinations and CEUS before surgery and at 1, 2, and 3 months after surgery. The intraneural enhancement pattern was calculated as an area under the curve (AUC) value in the entrapment site of the ulnar nerve within the cubital tunnel and in the area 1 cm proximal to the site (proximal site) at elbow flexion angles of 20° and 110°., Results: Serial electrophysiologic examinations showed improvements at 1, 2, and 3 months after surgery compared with before surgery. In the control group, the AUC values of the central part of the cubital tunnel and proximal sites showed no substantial changes with the increase in elbow flexion. In the CuTS group, the AUC in the proximal site at 110° of elbow flexion was decreased compared with that at 20° of flexion before surgery. The AUC values for both the entrapment and proximal sites at 20° and 110° of elbow flexion were the most increased at 2 months after surgery compared with before surgery., Conclusions: Increased elbow flexion in patients with CuTS influences the intraneural blood flow of the ulnar nerve. Surgery for CuTS alters the intraneural blood flow., Clinical Relevance: Quantitative evaluation of the intraneural blood flow of the ulnar nerve using CEUS may be a new supplementary diagnostic tool for CuTS and an indicator for the evaluation of postoperative recovery from nerve damage., (Copyright © 2022 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Introduction: The usefulness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests in asymptomatic individuals has not been well validated, although they have satisfied sensitivity and specificity in symptomatic patients. In this study, we investigated the significance of IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects., Methods: From June 2020, we recruited 10,039 participants to the project named the University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS), and measured iFlash-SARS-CoV-2 IgM and IgG (YHLO IgM and IgG) titers in the collected serum. For the samples with increased IgM or IgG titers, we performed additional measurements using Elecsys Anti-SARS-CoV-2 Ig (Roche total Ig) and Architect SARS-CoV-2 IgG (Abbott IgG) and investigated the reactivity to N, S1, and receptor binding domain (RBD) proteins., Results: After setting the cutoff value at 5 AU/mL, 61 (0.61%) were positive for YHLO IgM and 104 (1.04%) for YHLO IgG. Few samples with elevated YHLO IgM showed reactivity to S1 or RBD proteins, and IgG titers did not increase during the follow-up in any samples. The samples with elevated YHLO IgG consisted of two groups: one reacted to S1 or RBD proteins and the other did not, which was reflected in the results of Roche total Ig., Conclusions: In SARS-CoV-2 seroepidemiological studies of asymptomatic participants, sufficient attention should be given to the interpretation of the results of YHLO IgM and IgG, and the combined use of YHLO IgG and Roche total Ig might be more reliable., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Mitochondrial toxicity is an important factor to predict drug-induced liver injury (DILI). Previous studies have focused predominantly on mitochondrial toxicities due to parent forms, and no study has adequately evaluated metabolite-induced mitochondrial toxicity. Moreover, previous studies have used HepG2 cells, which lack many cytochrome P450 (CYP) genes. To overcome this problem, CYP-introduced HepG2 cells were constructed using several gene transfer technologies, including adenoviruses and plasmids. However, these methods only led to a transient expression of CYP genes. In the present study, usefulness of four CYPs introduced-HepG2 (TC-Hep) cells previously constructed through mammalian artificial chromosome technology were examined, especially from the perspective of mitochondrial toxicity. First, we evaluated the effects of known compounds, such as rotenone and flutamide, on mitochondrial toxicity and cell death in TC-Hep cells cultured in galactose conditions. Expectedly, rotenone-induced cell death ameliorated because rotenone was metabolized by CYPs into inactive form(s) and flutamide-induced cell death increased in TC-Hep cells. Second, we evaluated five compounds that caused liver injury in clinical phase and were discontinued during pharmaceutical development. The present in vitro tool suggested that three of the five compounds caused metabolite-induced mitochondrial toxicities. In conclusion, the present in vitro tool could easily and inexpensively detect metabolite-induced mitochondrial toxicity; hence, it can be useful for predicting DILI in preclinical phase., Competing Interests: Declarations of Competing Interest None., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Introduction: The worldwide pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to date. Given that some of the patients with coronavirus disease 2019 (COVID-19) are asymptomatic, antibody tests are useful to determine whether there is a previous infection with SARS-CoV-2. In this study, we measured IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects in The University of Tokyo, Japan., Methods: From June 2020, we recruited participants, who were students, staff, and faculty members of The University of Tokyo in the project named The University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS). Following blood sample collection, participants were required to answer an online questionnaire about their social and health information. We measured IgG and IgM titers against SARS-CoV-2 using iFlash-SARS-CoV-2 IgM and IgG detection kit which applies a chemiluminescent immunoassay (CLIA) for the qualitative detection., Results: There were 6609 volunteers in this study. After setting the cutoff value at 10 AU/mL, 32 (0.48%) were positive for IgG and 16 (0.24%) for IgM. Of six participants with a history of COVID-19, five were positive for IgG, whereas all were negative for IgM. The median titer of IgG was 0.40 AU/mL and 0.39 AU/mL for IgM. Both IgG and IgM titers were affected by gender, age, smoking status, and comorbidities., Conclusions: Positive rates of IgG and IgM titers were relatively low in our university. Serum levels of these antibodies were affected by several factors, which might affect the clinical course of COVID-19., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Purpose: Peri-coronary adipose tissue attenuation expressed by fat attenuation index (FAI) on coronary CT angiography (CCTA) reflects peri-coronary inflammation and is associated with cardiac mortality. We aimed to investigate the association between FAI and whole vessel and lesion plaque quantification on CCTA in stable patients with intermediate epicardial stenosis evaluated by fractional flow reserve (FFR)., Methods: A total of 187 left anterior descending arteries (LAD) with intermediate stenosis who underwent FFR measurement and CCTA were studied. FAI was assessed by the crude analysis of the mean CT attenuation value of LAD on CCTA. Determinants of FAI and FFR were explored. Furthermore, the impact of combined baseline data, CCTA-derived lesion plaque assessment, whole vessel quantification, cardiac mass and FAI on discrimination efficacy for ischemia was evaluated as FFR used for a reference standard., Results: The mean FAI and the median FFR values were -73.0 and 0.77, respectively. Multivariate analysis revealed that male, CCTA-derived positive remodeling, lower minimum lumen area, higher target vessel total cardiac mass, and lower FFR were independent predictors of FAI. CCTA-derived two-dimensional and three-dimensional analysis and FAI were independently and significantly associated with FFR values. Net reclassification index and integrated discrimination improvement index were both significantly improved when FAI was added to the baseline model for lesions with FFR <0.75, but not for FFR≤0.80., Conclusions: FAI was associated with FFR, CCTA-derived two-dimensional and three-dimensional lumen and plaque quantification and cardiac mass in patients with intermediate lesions in LAD, indicating that comprehensive CTA assessment may provide risk-stratification., Competing Interests: Declaration of competing interest All authors declare that there is no conflict of interest relevant to the submitted work., (Copyright © 2020 Society of Cardiovascular Computed Tomography. Published by Elsevier Inc. All rights reserved.)

The pleiotropic effects of statins, including an antiarthritic potential, have been noted. This study aimed to determine the efficacy of statins on rheumatoid arthritis (RA) and clarify how statins affect its pathogenesis. Fluvastatin (500 μg/kg/day) or vehicle was given per os to env-pX rats, which carry the human T-cell leukemia virus type I env-pX gene and spontaneously develop destructive arthritis mimicking RA, for 30 days. Blood sampling and ultrasonography (US) of the ankle joints were conducted on days 0, 10, 20, and 30. On day 30, all rats were euthanized, and the ankle joints were subjected to histological analysis. To clarify how fluvastatin affects the pathogenesis of RA, comprehensive serum exosomal microRNA (miRNA) analysis was performed. Gene expression in the primary culture of synovial fibroblasts derived from arthritic rat and human and non-arthritic rat periarticular tissues was determined quantitatively by real-time reverse transcription-polymerase chain reaction (RT-PCR). As a result, the development of arthritis in env-pX rats was significantly suppressed by fluvastatin, which was evident from the viewpoints of serology, US imaging, and histology. Comprehensive serum exosomal miRNA analysis suggested that the expression of Rho GTPase-activating protein 12 (Arhgap12) was decreased in arthritic env-pX rats but increased with the administration of fluvastatin. Corresponding results were obtained by quantitative RT- PCR using primary culture of synovial fibroblasts. The collective findings suggest that fluvastatin prevents the development of arthritis in env-pX rats via the up-regulation of ARHGAP12. This study suggests that ARHGAP12 can be a possible therapeutic target of RA., Competing Interests: Declaration of Competing Interest Tatsuya Atsumi received grant/research support from Takeda Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., AbbVie, Inc., Daiichi Sankyo Co., Ltd., Astellas Pharma, Inc., AYUMI Pharmaceutical Corp., Asahi Kasei Pharma Corporation, Eisai Co., Ltd., and Mitsubishi Tanabe Pharma Co. and has taken part in speakers' bureaus for Takeda Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., AbbVie, Inc., Bristol-Myers Squibb Co., Daiichi Sankyo Co., Ltd., Astellas Pharma, Inc., AYUMI Pharmaceutical Corp., UCB Japan Co., Ltd., Novartis Co., Janssen Pharmaceutical K.K., Asahi Kasei Pharma Corporation, Eisai Co., Ltd., Alexion, Inc., and Mitsubishi Tanabe Pharma Co. The other authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: There remains a residual risk for acute myocardial infarction (AMI) even with low low-density lipoprotein cholesterol (LDL-C) levels. This study aimed to characterize the culprit lesion morphology of AMI by optical coherence tomography (OCT) in patients with low LDL-C., Methods: Four-hundred and nine culprit lesions of 409 patients with their first presentation of AMI imaged by OCT were investigated. OCT analysis included the presence of plaque rupture and thin-capped fibroatheroma (TCFA). Fibrous cap thickness and lipid length were also measured. Intravascular ultrasound (IVUS) was performed in 368 (90.0%) patients. OCT and IVUS findings were compared between patients with LDL-C < 100 mg/dl (lower-LDL group) and those with LDL ≥ 100 mg/dl (higher-LDL group)., Results: Lower-LDL group included 93 (22.7%) patients. Plaque rupture (54.8% vs. 68.7%, p = 0.018) and TCFA (39.8% vs. 54.6%, p = 0.013) were less frequently observed in lower-LDL than in higher-LDL. Fibrous cap was thicker [73 (59-109) µm vs. 63 (57-83) µm, p = 0.028] and lipid length was smaller [5.4 (2.3-9.9) mm vs. 7.1 (4.1-10.5) mm, p = 0.012] in lower-LDL than in higher-LDL. There were no significant differences in IVUS parameters including plaque burden or remodeling index between the two groups., Conclusions: Patients with lower LDL-C showed more prevalent intact fibrous cap and less vulnerable features in the culprit lesions, which may suggest the need for exploring a specific strategy for the prevention of plaque erosion in low LDL-C subjects., (Copyright © 2020 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Background: The Psychosocial Assessment of Candidates for Transplantation (PACT) is a validated instrument for evaluating psychosocial risk factors in transplant candidates., Objectives: This study examined reliability and validity of the Japanese version of the PACT (J-PACT)., Methods: PACT is a clinician-rated scale consisting of an initial rating, 8 subscales, and a final rating. J-PACT was developed through a translation and back-translation procedure. Seventy adult patients who underwent allogeneic hematopoietic stem cell transplant between April 2009 and December 2013 received retrospective J-PACT ratings based on medical records. Interrater reliability and concurrent validity with Hospital Anxiety and Depression Scale (HADS) and Profile of Mood Status (POMS) scores were assessed., Results: Interrater reliability for each J-PACT item was generally high, ranging from 0.53 (drug and alcohol use)-0.93 (support stability). The concurrent validity analyses revealed the following significant relationships (p < 0.05). Higher support stability was associated with lower HADS depression (p = 0.02), POMS anger (p = 0.001), POMS fatigue (p = 0.03), and POMS confusion (p = 0.01) scores. Higher support availability was associated with lower POMS anger scores (p = 0.01). More suitable personality was associated with lower HADS anxiety (p = 0.04) and HADS depression (p = 0.048) scores. Better scores on lifestyle factors and alcohol use were both associated with lower POMS confusion scores (p = 0.01, 0.04, respectively). Better final rating was associated with lower HADS anxiety (p = 0.03) and HADS depression (p = 0.02) scores., Conclusion: J-PACT was reliable and valid, although further study is needed to confirm these findings., (Copyright © 2017 The Academy of Psychosomatic Medicine. Published by Elsevier Inc. All rights reserved.)

We previously demonstrated that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) was expressed in osteoblasts and participated in the regulation of bone homeostasis. However, it remains unclear how Ifrd1 expression itself is regulated in osteoblasts. In the present study, we investigated the upstream regulatory mechanisms of Ifrd1 in osteoblasts during osteoblastogenesis. Ifrd1 protein expression and runt-related transcription factor 2, the master regulator of osteoblastogenesis, were markedly upregulated by bone morphogenetic protein 2 (BMP-2) stimulation in primary osteoblasts. Moreover, BMP-2 stimulation significantly induced Ifrd1 mRNA expression and promoter activity in osteoblasts. LDN193189, an inhibitor of activin-like kinase 2/3, almost completely inhibited the BMP-2-induced increase in Ifrd1 protein expression. There were at least two putative Smad-binding elements in the 5'-flanking region, which was highly conserved between mouse and human Ifrd1 genes. Co-introduction of both Smad4 and Smad1 significantly increased Ifrd1 promoter activity in osteoblasts. In addition, BMP-2 induced the recruitment of Smad1 to the Ifrd1 promoter in osteoblasts. Moreover, BMP-2-dependent osteoblastogenesis was further enhanced in Ifrd1 knocked-down osteoblasts, as determined by the intensity of Alizarin red stain and marker gene expression. These results suggest that BMP-2 directly induces Ifrd1 expression at the transcriptional level in osteoblasts via the Smad pathway, and Ifrd1 negatively regulates BMP-2-dependent osteoblastogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Many studies have shown that cerebrospinal fluid (CSF) behaves irregularly, rather than with laminar flow, in the various CSF spaces. We adapted a modified previously known magnetic resonance imaging technique to visualize irregular CSF motion. Subsequently, we assessed the usefulness and clinical significance of the present method., Materials and Methods: Normal CSF motion in 10 healthy volunteers was visualized with the dynamic improved, motion-sensitized, driven-equilibrium steady-state free precession technique. Subsequently, CSF motion visualization with a modified sequence was applied to 3 patients., Results: In healthy volunteers, we achieved visualization of the irregularity of CSF flow in the ventricles and spinal canal, whereas CSF motion was diminished in the peripheral part of the intracranial subarachnoid space. In one case, we confirmed the patency of the patient's third ventriculostomy fenestration site. In the other, we verified the usefulness of the proposed sequence for determining the communication between the ventricle or subarachnoid space and the cyst., Conclusions: Using the present sequence, we obtained images that accentuated CSF motion, which is largely composed of irregular motion. This method does not require pulse triggering or complex post-processing of images and allows visualization of CSF motion in a short period of time in selected whole imaging planes. It can therefore be applied clinically to diagnose various diseases that cause abnormalities in the CSF space., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Bile acid (BA) retention within hepatocytes is an underlying mechanism of cholestatic drug-induced liver injury (DILI). We previously developed an assay using sandwich-cultured human hepatocytes (SCHHs) to evaluate drug-induced hepatocyte toxicity accompanying intracellular BA accumulation. However, due to shortcomings commonly associated with the use of primary human hepatocytes (e.g., limited availability, lot-to-lot variability, and high cost), we examined if the human hepatic stem cell line, HepaRG, might also be applicable to our assay system. Consequently, mRNA expression levels of human BA efflux and uptake transporters were lower in HepaRG cells than in SCHHs but higher than in HepG2 human hepatoma cells. Nevertheless, HepaRG cells and SCHHs showed similar toxicity responses to 22 selected drugs, including cyclosporine A (CsA). CsA (10 μM) was cytotoxic toward HepaRG cells in the presence of BAs and also reduced the biliary efflux rate of [(3)H]taurocholic acid from 38.5% to 19.2%. Therefore, HepaRG cells are useful for the evaluation of BA-dependent drug toxicity caused by biliary BA efflux inhibition. Regardless, the prediction accuracy for cholestatic DILI risk was poor for HepaRG cells versus SCHHs, suggesting that our DILI model system requires further improvements to increase the utility of HepaRG cells as a preclinical screening tool., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Objective: To determine whether low HDL-C is a risk factor for adverse cardiovascular events in patients with known CAD., Methods: We evaluated 10,391 patients who underwent PCI from January 2005 to December 2007. In total, 3838 (36.9%) patients had low HDL-C (HDL-C <40 mg/dL in males and <50 mg/dL in females) and 6553 (63.1%) patients had normal HDL-C based on measurements on admission., Results: The unadjusted 5-year incidence of major adverse cardiac events (MACE: composite of cardiovascular death, myocardial infarction or stroke) was significantly higher in the low HDL-C group than in the normal HDL-C group (17.6% vs. 14.0%, P < 0.0001). However, after adjusting for confounders, low HDL-C was not associated with a higher risk of MACE (adjusted hazard ratio [HR] 1.07, 95% confidence interval (CI) 0.97-1.19; P = 0.19). There was no significant interaction between the effect of low HDL-C on MACE and several subgroup factors including age, sex, clinical presentation of CAD, statins use, serum low-density lipoprotein cholesterol level, and serum triglycerides level., Conclusion: Low HDL-C, as compared with normal HDL-C, was not associated with higher 5-year risk of MACE in patients who underwent PCI., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Plant cells are sensitive to salinity stress and do not require sodium as an essential element for their growth and development. Saline soils reduce crop yields and limit available land. Research shows that HKT transporters provide a potent mechanism for mediating salt tolerance in plants. Knowledge of the molecular ion transport and regulation mechanisms and the control of HKT gene expression are crucial for understanding the mechanisms by which HKT transporters enhance crop performance under salinity stress. This review focuses on HKT transporters in monocot plants and in Arabidopsis as a dicot plant, as a guide to efforts toward improving salt tolerance of plants for increasing the production of crops and bioenergy feedstocks., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Atrioventricular (AV) block is one of the main clinical manifestations in patients with cardiac sarcoidosis (CS). Although steroid therapy is considered to be effective for AV block, the efficacy has not been demonstrated in detail., Methods and Results: Fifteen CS patients presenting with advanced or complete AV block were retrospectively investigated. All patients were treated with 30mg/day of prednisone after device implantation, which was tapered to a maintenance dosage of 5-10mg/day. During a mean follow-up of 7.1 years, AV block resolved to normal conduction or first-degree AV block in 7 patients (recovery group). The improvement was driven within the first week of steroid therapy in 4 patients, while 3 patients showed late recovery of AV conduction. The remaining 8 patients were classified as the non-recovery group. The recovery group showed a higher left ventricular ejection fraction (69.4±8.9% versus 44.1±19.3%, p=0.029) and higher prevalence of advanced AV block (87.5% versus 28.6%, p=0.040) compared with those of the non-recovery group. In patients with the recovery group, there was no late recurrence of AV block during the follow-up period., Conclusions: Early initiation of steroid therapy may be effective for AV block, and steroid therapy before device implantation is a possible therapeutic strategy for some selected patients., (Copyright © 2013 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Previously, we reported a long-lasting inhibition of transport mediated by organic anion-transporting polypeptides (OATPs) in humans and rats by cyclosporin A (CsA). In the present study, we examined the effects of several other compounds on OATP1B1-mediated transport, with a focus on long-lasting inhibition. Effects of coincubation, preincubation, or preincubation plus coincubation of 12 compounds on uptake of estrone 3-sulfate (E1 S) in OATP1B1-expressing HEK293T cells were examined. The OATP1B1 inhibitors used in the present study inhibited OATP1B1-mediated uptake of E1 S in a concentration-dependent manner. Among them, saquinavir and ritonavir in addition to CsA exhibited long-lasting inhibitory effects on OATP1B1-mediated transport of E1 S at ≥ 5 and 25 μM, respectively, even after they were washed out from the incubation buffer. After preincubation with saquinavir, its inhibitory effect on OATP1B1 remained for at least 6 h, whereas the effect of ritonavir did not remain. Protein expression of OATP1B1 was not altered by preincubation with 25 μM saquinavir or ritonavir. The present study firstly showed that saquinavir and ritonavir as well as CsA have long-lasting inhibitory effects on OATP1B1. But, at plasma unbound concentrations of saquinavir and ritonavir in clinical situations, they may not cause long-lasting inhibition of OATP1B1., (Copyright © 2013 Wiley Periodicals, Inc.)

Enhancers are flexible in terms of their location and orientation relative to the genes they regulate. However, little is known about whether the flexibility can be applied in every combination of enhancers and genes. Enhancer detection with transposable elements is a powerful method to identify enhancers in the genome and to create marker lines expressing fluorescent proteins in a tissue-specific manner. In the chordate Ciona intestinalis, this method has been established with a Tc1/mariner superfamily transposon Minos. Previously, we created the enhancer detection line E[MiTSAdTPOG]15 (E15) that specifically expresses green fluorescent protein (GFP) in the central nervous system (CNS) after metamorphosis. In this study, we identified the causal insertion site of the transgenic line. There are two genes flanking the causal insertion of the E15 line, and the genomic region around the insertion site contains the enhancers responsible for the expression in the endostyle and gut in addition to the CNS. We found that the endostyle and gut enhancers show sensitivity to the orientation of the GFP gene for their enhancer activity. Namely, the enhancers cannot enhance the expression of GFP which is inserted at the same orientation as the E15 line, while the enhancers can enhance GFP expression inserted at the opposite orientation. The CNS enhancer can enhance GFP expression in both orientations. The DNA element adjacent to the endostyle enhancer is responsible for the orientation sensitivity of the enhancer. The different sensitivity of the enhancers to the orientation of the transgene is a cause of CNS-specific GFP expression in the E15 line., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Vernonia amygdalina (VA), Carica papaya (CP), and Tapinanthus sessilifolius (ML) are widely used in some countries as medicinal herbs to treat ailments including malaria, cancer, and diabetes. We previously reported the inhibitory effects of these herbs on permeability glycoprotein (P-gp) in Caco-2 cell monolayers. This study used ex vivo and in vivo models to investigate the likelihood of P-gp-mediated herb-drug interactions occurring. The study utilized excised rat intestinal tissues mounted in Ussing chambers to predict changes in drug absorption and an in vivo study in rats using digoxin as the P-gp substrate. Apparent permeability values and pharmacokinetic parameters of digoxin were compared to determine if co-administration of digoxin with ML, CP, or VA modulated the activity of P-gp. When VA was co-administered, the total area under the plasma concentration-time curve was significantly higher (2.1-fold) than when digoxin was administered alone. Co-administration of ML, VA, and CP significantly increased the mean digoxin apparent permeability in the mucosal-to-serosal direction by 7.8, 43.3, and 54.5%, respectively, in comparison to when digoxin was administered alone. These findings suggest that VA increases intestinal absorption of digoxin in vivo by inhibiting P-gp and may also modulate the pharmacokinetic disposition of other p-gp substrate drugs.

Background: Chronic obstructive pulmonary disease (COPD) is a progressive condition that classically causes dyspnea during physical activity. Destruction of alveoli and bronchostenosis are thought to lead to shortness of breath and result in decreased physical activity. In this study, we examined the influence of inhaled procaterol on exercise therapy for pulmonary rehabilitation., Methods: Patients with moderate to severe stable COPD were randomly divided into 2 groups those who inhaled procaterol before exercise (n=10) and those who did not (control group) (n=11). For 12 weeks, all patients performed their pulmonary rehabilitation exercises at home. We measured the 6-minute walking distance (6MWD) to assess exercise tolerance and used St. George's respiratory questionnaire (SGRQ) to assess health-related quality of life (HRQOL) before and after the 12-week exercise program., Results: Compared to the control group, the group receiving inhaled procaterol showed significant improvement of 6MWD and SGRQ scores., Conclusion: Our data suggest that a pulmonary rehabilitation program combined with inhaled procaterol can improve both HRQOL and exercise tolerance in COPD patients., (Copyright © 2012 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

The purpose of the present study is to examine the long-lasting inhibition of intestinal organic anion transporting polypeptides (Oatps) by cyclosporin A (CsA) in rats using fexofenadine (FEX) as a probe drug. We examined the pharmacokinetics of FEX after its intravenous or oral administration to rats at 3 or 24 h after the oral administration of CsA. When FEX was administered at 3 h after the administration of CsA, its plasma concentration increased regardless of whether it was administered intravenously or orally. When FEX was intravenously administered at 24 h after the oral administration of CsA, its plasma concentration was increased; however, that observed after its oral administration was not significantly different from the vehicle-treated control. When FEX was administered at 3 h after the administration of CsA, the hepatic availability (F(h)) and the fraction absorbed in the intestine as an unchanged form (F(a)·F(g)) of FEX were increased, resulting in increased bioavailability (=F(a)·F(g)·F(h)). At 24 h after the administration of CsA, the F(h) of FEX was increased, whereas its bioavailability was decreased, suggesting that its F(a)·F(g) was decreased because of the long-lasting inhibition. In conclusion, CsA has long-lasting inhibitory effects on Oatps in the rat intestine as well as in the liver., (Copyright © 2012 Wiley Periodicals, Inc.)

Cyclosporin A (CsA) causes a number of clinically relevant drug-drug interactions (DDIs) by inhibiting OATP1B1 and OATP1B3. In the present study, long-lasting inhibitory effects of CsA on these transporters were examined in comparison to tacrolimus (TCR). OATP1B1- and OATP1B3-expressing HEK293T cells, OATP1B1-expressing MDCK II cells, and human hepatocytes were preincubated with CsA or TCR, and uptake studies were carried out in their presence or absence. Western blot or immunohistochemical studies were done in OATP1B1-expressing HEK293T cells. The pretreatment of OATP1B1- and OATP1B3-expressing cells with 0.5-10 µM CsA, but not TCR, resulted in a reduction in their activity, even after washing out CsA from the incubation media. Preincubating the cells with CsA significantly enhanced its inhibitory effects on OATP1B1 and OATP1B3 by coincubation at 0.1-1 µM. Preincubation with 1 µM CsA caused a reduction in OATP1B1 activity for at least 18 h after its removal. The expression of OATP1B1 was not affected by incubation with CsA and no obvious change in its intracellular localization was observed. The long-lasting inhibition by CsA was also observed in human hepatocytes. Thus, CsA has a long-lasting inhibitory effect on OATP1B1 and OATP1B3. It may attribute to the clinically relevant DDIs between OATP substrates and CsA.

Drug-induced liver injury (DILI) is a major reason for the dropout of candidate compounds from drug testing and the withdrawal of pharmaceuticals from clinical use. Among the various mechanisms of liver injury, the accumulation of bile acids (BAs) within hepatocytes is thought to be a primary mechanism for the development of DILI. Although bile salt export pump (BSEP) dysfunction is considered a susceptibility factor for DILI, little is known about the relationship between drug-induced BSEP dysfunction and BA-dependent hepatotoxicity. Furthermore, few methods are at hand for the systematic and quantitative evaluation of BA-dependent DILI. This study aimed to construct a model of DILI by employing sandwich-cultured hepatocytes (SCHs). SCHs can be used to assess functions of canalicular transporters such as BSEP and the activity of metabolic enzymes. Here, the impact of 26 test compounds (ritonavir, troglitazone, etc.) was investigated on BA-dependent cytotoxicity in SCHs. SCHs were exposed to each compound for 24h with or without BAs (glycochenodeoxycholic acid, deoxycholic acid, etc.). As a result, BA-dependent toxicity was observed for 11 test compounds in SCHs treated in the presence of BAs, while no signs of toxicity were observed for SCHs treated in the absence of BAs. Of the 11 compounds, nine were known BSEP inhibitors. Moreover, for some compounds, an increase in the severity of BA-dependent toxicity was observed in SCHs that were co-treated with 1-aminobenzotriazole, a non-selective inhibitor of cytochrome P450 (CYP450)-mediated drug metabolism. These results indicate that the SCH-based model is likely to prove useful for the evaluation of BA-dependent DILI, including the effects of drug metabolism and BSEP inhibition on liver injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Little is known about the tachyarrhythmias relating to respiration. Case reports presented patients with respiratory cycle-dependent atrial tachycardias (RCATs), which cyclically emerge after starting inspiration and cease during expiration., Objective: The aim of the present study was to elucidate the prevalence, characteristics, and long-term outcome after radiofrequency catheter ablation (RFCA) of RCATs., Methods: The electrocardiographic and electrophysiologic properties and results of RFCA were analyzed in 60 patients with a total of 71 focal atrial tachycardias (ATs)., Results: Nine RCATs (13%) were observed in 7 patients (12%). RCATs were irregular, with a mean cycle length ranging from 220 to 650 ms, and developed incessantly accounting for 32% ± 14% of the 24-hour heartbeats. The P-wave morphology was positive or biphasic (positive to negative) in V1, and positive in I and II. The electroanatomical mapping demonstrated a centrifugal activation pattern, with the earliest site located at the antrum of the right superior pulmonary vein (RSPV), inside the RSPV, and inside the superior vena cava (SVC) in 4, 2, and 3 RCATs, respectively. Radiofrequency energy delivery at the earliest site or the electrical isolation of the RSPV and SVC suppressed all RCATs. During a follow-up of 25 ± 15 months, 1 RCAT recurred and was eliminated in a second procedure., Conclusion: RCATs were observed in 13% of the focal ATs. As presumed from the P-wave morphologies, their foci converged around the RSPV or inside the SVC. RFCA was effective to eliminate RCATs., (Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Despite containing only approximately 330 cells, the central nervous system (CNS) of Ciona intestinalis larvae has an architecture that is similar to the vertebrate CNS. Although only vertebrates have a distinct hypothalamus-the source of numerous neurohormone peptides that play pivotal roles in the development, function, and maintenance of various neuronal and endocrine systems, it is suggested that the Ciona brain contains a region that corresponds to the vertebrate hypothalamus. To identify genes expressed in the brain, we isolated brain vesicles using transgenic embryos carrying Ci-β-tubulin(promoter)::Kaede, which resulted in robust Kaede expression in the larval CNS. The associated transcriptome was investigated using microarray analysis. We identified 565 genes that were preferentially expressed in the larval brain. Among these genes, 11 encoded neurohormone peptides including such hypothalamic peptides as gonadotropin-releasing hormone and oxytocin/vasopressin. Six of the identified peptide genes had not been previously described. We also found that genes encoding receptors for some of the peptides were expressed in the brain. Interestingly, whole-mount in situ hybridization showed that most of the peptide genes were expressed in the ventral brain. This catalog of the genes expressed in the larval brain should help elucidate the evolution, development, and functioning of the chordate brain., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We evaluated a novel three-dimensional primary culture system using micro-space plates to determine the expression levels of 61 target (drug-metabolizing enzymes, transporters, and nuclear receptors) mRNAs in human hepatocytes. We measured mRNA expression levels of many target genes in four lots of cryopreserved human hepatocyte primary cells after 120 h of culture and compared differences in mRNA expression levels between cultures using traditional plates and those using micro-space plates. In this study, we show that the mRNA levels of many experimental targets in human hepatocytes before inoculation resemble the levels inside the human liver. Furthermore, we show that the rate of change of expression levels of many target mRNAs relative to the value before inoculation of the hepatocytes into micro-space plates was relatively smaller than the rate of change in hepatocytes inoculated into traditional plates. Pharmacokinetics-related examinations using this system are possible within a time frame of 120 h. We report that this novel three-dimensional culture system reproduces mRNA expression levels that are nearer to those in the liver in vivo and is an excellent platform for maintaining mRNA expression levels of drug-metabolizing enzymes and transporters when compared to common monolayer cultures.

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5h following LPS treatment (100microg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10microM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices., (Copyright 2010 Elsevier Inc. All rights reserved.)

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is a receptor for oxidized LDL, and is strongly expressed in endothelial cells at an early stage of atherosclerosis. LOX-1 expression in adipocytes is induced by PPARgamma (ligands and appears to be involved in adipocyte cholesterol metabolism. However, the role of adipose tissue LOX-1 in high-fat diet-induced obesity is unknown. We found that mRNA levels of adipose tissue LOX-1 were markedly increased in obese mice fed a high-fat diet (HFD) compared with those fed normal chow. The levels were closely correlated with those of a proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Then, LOX-1 knockout (LOX-1-KO) and wild-type (WT) mice were fed HFD for 16weeks. HFD feeding increased the body and mesenteric fat weights similarly in WT and LOX-1-KO mice. HFD-induced expressions of proinflammatory cytokines such as MCP-1, MIP-1alpha, and IL-6 were significantly less in LOX-1-KO than WT mice. Thus, LOX-1 is required for the HFD-induced expression of proinflammatory cytokines in the adipose tissue of obese mice., (Copyright 2010 Elsevier Inc. All rights reserved.)

Salicylic acid is a widely used nonsteroidal anti-inflammatory drug (NSAID). But it is known to cause serious liver damage occasionally. Mitochondrial dysfunction and oxidative stress are predicted to be the major factors of salicylic acid-induced liver injury. We investigated the influence of salicylic acid on ATP contents, oxygen consumption and lipid peroxidation in the presence of the same concentration of salicylic acid. Leakage of lactate dehydrogenase (LDH) was significantly higher in the presence of 5mM salicylic acid than in its absence. Salicylic acid-induced thiobarbituric acid-reactive substance (TBARS) formation and spontaneous chemiluminescence (CL) in rat hepatocytes, whereas antioxidants, such as promethazine (PMZ) and N,N-diphenylphenylenediamine (DPPD), suppressed both TBARS formation and LDH leakage. TBARS formation in rat liver microsomes was suppressed by diethyldithiocarbamate (a specific inhibitor of cytochrome P450 (CYP)2E1) and diclofenac (a specific inhibitor of CYP2C11). These results suggest that salicylic acid-induced lipid peroxidation was related to oxidative metabolism mediated by CYP2E1 and CYP2C11. On the other hand, 5mM salicylic acid induced a drastic decrease of ATP contents in rat isolated hepatocytes. Furthermore, mitochondrial respiration control ratio (RC ratio), calculated by State 3/State 4 also decreased with the increase of salicylic acid concentration. These findings suggest that salicylic acid would trigger mitochondrial dysfunction and cause ATP decrease, leading to lethal liver cell injury by lipid peroxidation, although this hypothesis remains to be elucidated in vivo., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Background: Many studies have described the clinical effects of treating critical limb ischemia with granulocyte colony-stimulating factor-mobilized autologous peripheral blood mononuclear cells (M-PBMNC); however, there are no long-term data available on survival, limb salvage, or prognostic factors., Methods: To investigate the long-term clinical outcomes of M-PBMNC implantation, we reviewed data for 162 consecutive patients with limb ischemia who were treated with M-PBMNC implantation at 6 hospitals between 2001 and 2006. A subset of 123 patients with homogenous clinical profiles was selected for prognostic factor analysis., Results: Of the 162 patients, 50 died during the follow-up period. The median follow-up time for surviving patients was 26.4 months. The 2-year survival rate was 65% for the 140 patients with arteriosclerosis obliterans (ASO), and 100% for the 11, 4 and 7 patients with thromboangiitis obliterans (TAO), diabetic gangrene (DG) and connective tissue disease (CTD), respectively. The 1-year amputation-free rates for ASO, TAO, DG and CTD were 70%, 79%, 75% and 83%, respectively. Common serious adverse events included heart failure (15 cases), myocardial infarction (15 cases), serious infection (13 cases), stroke (10 cases), and malignant tumor (9 cases). Significant negative prognostic factors associated with overall survival were ischemic heart disease and collection of a small number of CD34-positive cells. Factors associated with time-to-amputation and amputation-free survival were a combination of Fontaine classification and lower limb gangrene, and history of dialysis., Conclusions: Collection of a small number of CD34-positive cells and ischemic heart disease were associated with a reduction in overall survival., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

In the pathogenesis of doxorubicin (DXR)-induced cardiomyopathy, oxidative stress appears to play an important role. It has been reported that pentosidine and N(epsilon)-(carboxymethyl)lysine (CML), advanced glycation end-products (AGEs), are formed by the combined processes of glycation and oxidation and play a significant role in the process of complications of diabetic mellitus. We investigated the potential involvement of AGE formation in DXR-induced cardiomyopathy in rats. Male Crl:CD(SD) rats received intravenous injection of DXR at 2mg/kg or saline once weekly for 8 weeks, with or without daily treatment with the AGE formation inhibitors, aminoguanidine (AG, 25 mg/kg/day, i.p.) and pyridoxamine (PM, 60 mg/kg/day, i.p.). Time-course experiments revealed significantly increased pentosidine and CML in the heart in the DXR group from Week 6. These findings coincided with a decrease in fractional shortening (FS), an index of cardiac function, and the development of cardiomyopathy characterized by vacuolated hypertrophic myocardial fibers. There was a significant correlation between the myocardial AGEs and FS or plasma cardiac troponin-I. Immunohistochemical staining showed localization of pentosidine to the cytoplasm of vacuolated myocardial cells. In DXR-treated rats, oxidative stress was enhanced prior to any observed increase in pentosidine and CML levels in the heart. Hyperglycemia was not observed throughout the study period. Intervention by AG or PM treatment ameliorated the functional and morphological changes induced by DXR in the heart, in addition to lowered myocardial pentosidine and CML levels. These results suggested that DXR accelerates the formation of pentosidine and CML in the heart through enhanced oxidative stress and that AGE formation is involved in DXR-induced cardiomyopathy. The findings may enable development of novel preventive therapies and predictive biomarkers of DXR-induced cardiomyopathy., (2009 Elsevier Ireland Ltd. All rights reserved.)

The formation of morphine-3-glucuronide (M-3-G, pharmacologically inactive) and morphine-6-glucuronide (M-6-G, active metabolite) by liver microsomes from humans and rodents, including chimeric mice carrying human liver, was evaluated in the presence of fatty acyl-CoAs. Medium- to long-chain fatty acyl-CoAs, including oleoyl-CoAs, at a physiologic level (around 15 microM) markedly enhanced M-3-G formation catalyzed by rat liver microsomes. A separate experiment indicated that 15 microM oleoyl-CoA enhanced (14)C-UDP-glucuronic acid (UDPGA) uptake by microsomes. The activation by acyl-CoAs disappeared or was greatly reduced by either pre-treating microsomes with detergent or freezing/thawing the rat liver before preparation. Many of the microsomes prepared from frozen human livers (N=14) resisted oleoyl-CoA-mediated activation of UDP-glucuronosyltransferase (UGT) activity, including M-6-G formation, which is highly specific to humans. In sharp contrast, the activity of M-6-G and M-3-G formation in freshly-prepared hepatic microsomes from chimeric mice with humanized liver was potently activated by oleoyl-CoA. Thus, acyl-CoAs activate morphine glucuronidation mediated by human as well as rat UGTs. This activation is assumed to be due to the acyl-CoA-facilitated transportation of UDPGA, and microsomes need to maintain the intact conditions required for the activation. The function of UGT appears to be dynamically changed depending on the cellular acyl-CoA level in many species.

We evaluated a novel primary three-dimensional culture system for human hepatocytes using micro-space plates. The functional activity of human hepatocytes in primary culture was determined by measuring albumin secretion from hepatocytes to medium and measuring expression levels of albumin, CYP1A2 and CYP3A4 mRNA. Albumin secretion was higher in micro-space plates compared with traditional plates after 72 h of culture; the levels of albumin secretion from hepatocytes to medium in culture using micro-space plates after 96 h of culture were 2.7-fold higher than those in culture using traditional plates, and secretion of albumin in micro-space plate culture subsequently remained constant. Expression levels of albumin, CYP1A2 and CYP3A4 mRNA in the culture of hepatocytes were significantly higher using micro-space plates than using traditional plates. The inducibility of CYP1A2 and CYP3A4 mRNA after exposure to inducers in hepatocyte culture on micro-space plates was comparable to that in culture on traditional plates, while expression of CYP1A2 and CYP3A4 mRNA after exposure to inducers was higher on micro-space plates than on traditional plates. The present study demonstrates that a novel primary three-dimensional culture system of cryopreserved human hepatocytes using micro-space plates could be used for evaluating the induction of drug-metabolizing enzymes in humans. This in vitro method may thus be useful for screening the induction potency of new drug candidates.

Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9+0 structure similar to vertebrate primary cilia, a 9+0 structure with electron-dense materials in the center, a 9+2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan.

GLUT4 shows decreased levels in failing human adult hearts. We speculated that GLUT4 expression in cardiac muscle may be fine-tuned by microRNAs. Forced expression of miR-133 decreased GLUT4 expression and reduced insulin-mediated glucose uptake in cardiomyocytes. A computational miRNA target prediction algorithm showed that KLF15 is one of the targets of miR-133. It was confirmed that over-expression of miR-133 reduced the protein level of KLF15, which reduced the level of the downstream target GLUT4. Cardiac myocytes infected with lenti-decoy, in which the 3'UTR with tandem sequences complementary to miR-133 was linked to the luciferase reporter gene, had decreased miR-133 levels and increased levels of GLUT4. The expression levels of KLF15 and GLUT4 were decreased at the left ventricular hypertrophy and congestive heart failure stage in a rat model. The present results indicated that miR-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiomyocytes.

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic "skeleton" essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal-epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.

Inflammation is an important pathophysiological event in drug-induced liver injury, which is subsequent to metabolic activation and covalent binding of the reactive metabolites to target proteins. Cytokines are recognized as pro- and anti-inflammatory mediators involved in the progression and regression of the toxicity. We thus hypothesized that disturbed balance of Th1/Th2 cytokines exacerbated the drug-induced hepatotoxicity. Acetaminophen-induced liver injury was investigated in two mouse strains, C57BL/6 and BALB/c, which develop predominantly Th1 and Th2 responses, respectively. More severe liver injury after intraperitoneal administration of acetaminophen was observed in C57BL/6 mice than in BALB/c mice. There was no strain difference in metabolism of acetaminophen into its reactive metabolite, N-acetyl-p-benzoquinone imine, which was assessed by early glutathione consumption. Liver mRNA expression of tumor necrosis factor-alpha (TNF-alpha) and IL-6 were measured as pro- and anti-inflammatory cytokines, respectively. TNF-alpha was highly induced 24 h after administration of acetaminophen in C57BL/6 mice, whereas no change in BALB/c mice. On the other hand, liver IL-6 mRNA expression in BALB/c mice was higher than C57BL/6 mice 24 h after the administration. In addition, treatment of CD-1 mice, another susceptible strain, with an anti-inflammatory polyphenol, resveratrol, protected mice against the acetaminophen-induced liver injury, and the mice with attenuated toxicity revealed lower expression of TNF-alpha and higher expression of IL-6. It is therefore suggested that acetaminophen-induced liver injury is associated with Th1-dominant response in Th1/Th2 cytokine balance, and TNF-alpha may play a pathological role in the toxicity.

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.

Chimeric mice, constructed by transplanting human hepatocytes, are useful for predicting the human metabolism of drug candidates. In this study, we investigated whether these mice show similar metabolic profile to humans by examining the hydroxylation of S-warfarin reported to be mainly metabolized to S-7-hydroxywarfarin (7-OH-warfarin), catalyzed by CYP2C9, in humans. When S-(3)H-warfarin was administered to chimeric mice and control (uPA(+/+)/SCID(wt/wt)) mice, the blood concentration-time curve was higher in chimeric than control mice. Plasma protein binding of S-(3)H-warfarin of chimeric and control mice amounted to 98.1 and 92.1%, respectively. When S-(3)H-warfarin was administered to these mice, radioactivity was mainly recovered in urine (81.7% in chimeric mice and 65.9% in control mice). After S-(3)H-warfarin was administered to these mice, the radioactivity was recovered in the bile of chimeric and control mice at 5.1 and 17.9%, respectively. The main urinary metabolite in chimeric mice was 7-OH-warfarin. the main urinary metabolite in control mice was S-4'-hydroxywarfarin. These results show that mass balance, metabolic disposition of S-(3)H-warfarin in chimeric mice with humanized liver were similar to reported human data.

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. miR-143 has been proposed to play a role in the differentiation of adipocytes in culture. However, the mechanism regulating the expression of miR-143 in adult adipose tissue during the development of obesity in vivo is unknown. Here in, we showed that the expression of miR-143 in the mesenteric fat was up-regulated in mice fed a high-fat diet. Increased miR-143 expression was associated with an elevated body weight and mesenteric fat weight. Furthermore, miR-143 levels were closely correlated with expression levels of adipocyte differentiation markers such as PPARgamma and aP2 as well as plasma levels of leptin, one of the important adipocytokines involved in insulin resistance. These findings provide the first evidence for the up-regulated expression of miR-143 in the mesenteric fat of high-fat diet-induced obese mice, which might contribute to the regulated expression of adipocyte genes involved in the pathophysiology of obesity.

The accumulation of visceral adipose tissue is closely associated with insulin resistance and metabolic syndrome. Therefore, it is important to identify genes that are required for adipocyte differentiation. To identify genes that are required for the differentiation of 3T3-L1 preadipocytes into mature adipocytes, we used retrovirus insertion-mediated random mutagenesis to generate 3T3-L1 cell lines that lose their ability to differentiate into mature adipocytes. One of the genes identified was TG-interacting factor (TGIF), a DNA binding homeodomain protein that has been demonstrated to suppress Smad-mediated activation of transforming growth factor beta (TGF-beta)-regulated transcription. In the TGIF-disrupted clone of 3T3-L1 preadipocytes, the rate of differentiation into mature adipocytes was clearly reduced compared with that in the wild-type clone. Suppression of TGIF by lentivirus-mediated RNAi also inhibited the differentiation of 3T3-L1 cells. Insulin specifically increased the abundance of TGIF protein, primarily by enhancing its stability. In addition, insulin caused the rapid accumulation of TGIF in the nuclei. Forced expression of exogenous TGIF repressed both endogenous and overexpressed Smad2/3-mediated promoter activity in 3T3-L1. These findings suggest that insulin specifically antagonizes TGF-beta signaling in preadipocytes by stabilizing the putative Smad transcriptional corepressor TGIF and regulates adipocyte differentiation.

Coumarin 7-hydroxylation (COH), which is catalyzed almost solely by human CYP2A6 and mouse CYP2A5, shows large differences in activity (humans>>mice) and inhibitor specificity between mice and humans. To differentiate human and mouse liver functions of chimeric mice (CM1, CM2 and CM3) prepared with hepatocytes from 3 donors, the microsomal COH activities were measured with and without benzaldehyde and undecanoic gamma-lactone as a specific inhibitor of human CYP2A6 and mice CYP2A5, respectively. The replacement % to human hepatocytes designated as replacement index (RI) was calculated from human specific cytokeratin 8/18 expression in the liver section. The COH activities correlated well with RIs in CM2 (R(2)=0.98) and CM3 (R(2)=0.94), except CM1 whose genotype of donor is CYP2A6*4/*4. However, the COH activities expressed as % of donor activities were not always coincident with RIs, and the inhibition pattern of CM2 and CM3 was human-type after RI exceeded approximately 50%. Subsequently, our attempts to use % of COH activities or inhibition patterns as an accurate functional replacement index were unsuccessful. Since the detection of human CYP2A6 protein in the liver and the steep increase of human albumin (hAlb) levels in the blood were begun from almost RI=50% similarly to the changes of inhibition pattern, RI=50% is the turning point for chimeric mice to have humanized liver function.