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1.  Prognostic value of cardiovascular magnetic resonance derived right ventricular function in patients with interstitial lung disease 
Cardiovascular magnetic resonance (CMR) provides non-invasive and more accurate assessment of right ventricular (RV) function in comparison to echocardiography. Recent study demonstrated that assessment of RV function by echocardiography was an independent predictor for mortality in patients with interstitial lung disease (ILD). The purpose of this study was to determine the prognostic significance of CMR derived RV ejection fraction (RVEF) in ILD patients.
We enrolled 76 patients with ILD and 24 controls in the current study. By using 1.5 T CMR scanner equipped with 32 channel cardiac coils, we performed steady-state free precession cine CMR to assess the RVEF. RV systolic dysfunction (RVSD) was defined as RVEF ≤45.0% calculated by long axis slices. Pulmonary hypertension (PH) was defined as mean pulmonary artery pressure (mPAP) of more than 25 mmHg at rest in the setting of pulmonary capillary wedge pressure ≤15 mmHg.
The median RVEF was 59.2% in controls (n = 24), 53.8% in ILD patients without PH (n = 42) and 43.1% in ILD patients with PH (n = 13) (p < 0.001 by one-way ANOVA). During a mean follow-up of 386 days, 18 patients with RVSD had 11 severe events (3 deaths, 3 right heart failure, 3 exacerbation of dyspnea requiring oxygen, 2 pneumonia requiring hospitalization). In contrast, only 2 exacerbation of dyspnea requiring oxygen were observed in 58 patients without RVSD. Multivariate Cox regression analysis showed that RVEF independently predicted future events, after adjusting for age, sex and RVFAC by echocardiography (hazard ratio: 0.889, 95% confidence interval: 0.809 – 0.976, p = 0.014).
The current study demonstrated that RVSD in ILD patients can be clearly detected by cine CMR. Importantly, low prevalence of PH (17%) indicated that population included many mild ILD patients. CMR derived RVEF might be useful for the risk stratification and clinical management of ILD patients.
PMCID: PMC4323037
Interstitial lung disease; Magnetic resonance imaging; Right ventricular function
2.  The selective tRNA aminoacylation mechanism based on a single G•U pair 
Nature  2014;510(7506):507-511.
Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNAAla selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNAAla with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3′-CCA region. Thus, the 3′-CCA region of tRNAAla with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the “non-reactive route”. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.
PMCID: PMC4323281  PMID: 24919148
3.  Amino acids inhibit kynurenic acid formation via suppression of kynurenine uptake or kynurenic acid synthesis in rat brain in vitro 
SpringerPlus  2015;4:48.
The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor at endogenous brain concentrations. Recent studies have suggested that increase of brain KYNA levels is involved in psychiatric disorders such as schizophrenia and depression. KYNA-producing enzymes have broad substrate specificity for amino acids, and brain uptake of kynurenine (KYN), the immediate precursor of KYNA, is via large neutral amino acid transporters (LAT). In the present study, to find out amino acids with the potential to suppress KYNA production, we comprehensively investigated the effects of proteinogenic amino acids on KYNA formation and KYN uptake in rat brain in vitro. Cortical slices of rat brain were incubated for 2 h in Krebs-Ringer buffer containing a physiological concentration of KYN with individual amino acids. Ten out of 19 amino acids (specifically, leucine, isoleucine, phenylalanine, methionine, tyrosine, alanine, cysteine, glutamine, glutamate, and aspartate) significantly reduced KYNA formation at 1 mmol/L. These amino acids showed inhibitory effects in a dose-dependent manner, and partially inhibited KYNA production at physiological concentrations. Leucine, isoleucine, methionine, phenylalanine, and tyrosine, all LAT substrates, also reduced tissue KYN concentrations in a dose-dependent manner, with their inhibitory rates for KYN uptake significantly correlated with KYNA formation. These results suggest that five LAT substrates inhibit KYNA formation via blockade of KYN transport, while the other amino acids act via blockade of the KYNA synthesis reaction in brain. Amino acids can be a good tool to modulate brain function by manipulation of KYNA formation in the brain. This approach may be useful in the treatment and prevention of neurological and psychiatric diseases associated with increased KYNA levels.
PMCID: PMC4318830
Amino acids; Kynurenic acid; Kynurenine; α7 nicotinic acetylcholine receptor; Dopamine; Neuropsychiatric disorders
4.  Impact of discontinuing non-steroidal antiinflammatory drugs on long-term recurrence in colonic diverticular bleeding 
AIM: To determine the effect of discontinuing non-steroidal antiinflammatory drugs (NSAIDs) on recurrence in long-term follow-up patients with colonic diverticular bleeding (CDB).
METHODS: A cohort of 132 patients hospitalized for CDB examined by colonoscopy was prospectively enrolled. Comorbidities, lifestyle, and medications (NSAIDs, low-dose aspirin, antiplatelet agents, anticoagulants, acetaminophen, and corticosteroids) were assessed. After discharge, patients were requested to visit the hospital on scheduled days during the follow-up period. The Kaplan-Meier method was used to estimate recurrence.
RESULTS: Median follow-up was 15 mo. The probability of recurrence at 1, 6, 12, and 24 mo was 3.1%, 19%, 27%, and 38%, respectively. Of the 41 NSAID users on admission, 26 (63%) discontinued NSAID use at discharge. Many of the patients who could discontinue NSAIDs were intermittent users, and could be switched to alternative therapies, such as acetaminophen or an antiinflammatory analgesic plaster. The probability of recurrence at 12 mo was 9.4% in discontinuing NSAID users compared with 77% in continuing users (P < 0.01, log-rank test). The hazard ratio for recurrence in the discontinuing NSAIDs users was 0.06 after adjusting for age > 70 years, right-sided diverticula, history of hypertension, and hemodialysis. No patients developed cerebrocardiovascular events during follow-up.
CONCLUSION: There is a substantial recurrence rate after discharge among patients hospitalized for diverticular bleeding. Discontinuation of NSAIDs is an effective preventive measure against recurrence. This study provides new information on risk reduction strategies for diverticular bleeding.
PMCID: PMC4306175  PMID: 25632204
Non-steroidal anti-inflammatories; Drug withdrawal; Diverticular hemorrhage; Hemodialysis; Antithrombotic drugs
5.  Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active site formation and catalytic specificity 
Journal of molecular biology  2014;426(8):1723-1735.
The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins, and is synthesized on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec, formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate (PLP)-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer-pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions, and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site, and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I PLP-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.
PMCID: PMC4306587  PMID: 24456689
Fold-type-I PLP-dependent enzyme; selenium metabolism; tRNASec; pentamer of dimers; 21st amino acid
6.  A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNACys 
The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu (“EF-Pyl”). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture. The MM1309 structures resemble those of bacterial/archaeal SelB, bacterial EF-Tu in the GTP-bound form, and archaeal initiation factor aIF2γ, in this order. The GMPPNP and GDP molecules are visible in their co-crystal structures. Isothermal titration calorimetry measurements of MM1309·GTP·Mg2+, MM1309·GDP·Mg2+, and MM1309·GMPPNP·Mg2+ provided dissociation constants of 0.43, 26.2, and 222.2 μM, respectively. Therefore, the affinities of MM1309 for GTP and GDP are similar to those of SelB rather than those of EF-Tu. Furthermore, the switch I and II regions of MM1309 are involved in domain–domain interactions, rather than nucleotide binding. The putative binding pocket for the aminoacyl moiety on MM1309 is too small to accommodate the pyrrolysyl moiety, based on a comparison of the present MM1309 structures with that of the EF-Tu·GMPPNP·aminoacyl-tRNA ternary complex. A hydrolysis protection assay revealed that MM1309 binds cysteinyl (Cys)-tRNACys and protects the aminoacyl bond from non-enzymatic hydrolysis. Therefore, we propose that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNACys.
PMCID: PMC4329189  PMID: 25618148
Crystal structure; Translation factor; GTP; tRNA
7.  NMR Studies of Thermo-responsive Behavior of an Amphiphilic Poly(asparagine) Derivative in Water 
Polymer  2014;55(1):278-286.
The thermo-responsive behavior of a unique biocompatible polymer, poly(N-substituted α/β-asparagine) derivative (PAD), has been studied with several NMR methods. The 1H and 13C solution NMR measurements of the PAD in DMSO-d6 were used to investigate the isolated polymer and perform spectral assignments. By systematic addition of D2O we have tracked structural changes due to aggregation and observed contraction of hydrophilic side chains. Solution and cross polarization / magic angle spinning (CP/MAS) 13C NMR approaches were implemented to investigate the aggregates of the PAD aqueous solution during the liquid to gel transition as the temperature was increased. At temperatures near 20 °C, all of the peaks from the PAD were observed in the 13C CP/MAS and 13C solution NMR spectra, indicating the presence of polymer chain nodes. Increasing the temperature to 40 °C resulted in a partial disentanglement of the nodes due to thermal agitation and further heating resulted in little to no additional structural changes. Deuterium T1–T2 and T2–T2 two-dimensional relaxation spectroscopies using an inverse Laplace transform, were also implemented to monitor the water–PAD interaction during the phase transition. At temperatures near 20 °C the dynamical characteristics of water were manifested into one peak in the deuterium T1–T2 map. Increasing the temperature to 40 °C resulted in several distinguishable reservoirs of water with different dynamical characteristics. The observation of several reservoirs of water at the temperature of gel formation at 40 °C is consistent with a physical picture of a gel involving a network of interconnected polymer chains trapping a fluid. Further increase in temperature to 70 °C resulted in two non-exchanging water reservoirs probed by deuterium T2–T2 measurements.
PMCID: PMC4299659  PMID: 25614708
8.  A 45,X/46,XY DSD (Disorder of Sexual Development) case with an extremely uneven distribution of 46,XY cells between lymphocytes and gonads 
In 45,X/46,XY DSDs, the proportion of the two cell lineages is uneven in different organs and tissues, and 45,X and 46,XY cells can be found throughout the body. The gonadal development of 45,X/46,XY patients depends on the population of 46,XY cells in the gonads and the clinical features are variable. We had a 45,X/46,XY DSD patient whose 46,XY population in peripheral blood was extremely low, less than 0.2%, and was not detected by FISH analysis. However, the patient showed bilateral testicular development and more than 50% of the cells in the gonads had the 46,XY karyotype. This case suggests that a drastically imbalanced distribution could occur in 45,X/46,XY DSD cases.
PMCID: PMC4322288
45,X/46,XY DSD (disorder of sexual development); mosaicism; chimerism; SRY; FISH (fluorescence in situ hybridization)
9.  Applicability of Preoperative Nuclear Morphometry to Evaluating Risk for Cervical Lymph Node Metastasis in Oral Squamous Cell Carcinoma 
PLoS ONE  2014;9(12):e116452.
We previously reported the utility of preoperative nuclear morphometry for evaluating risk for cervical lymph node metastases in tongue squamous cell carcinoma. The risk for lymph node metastasis in oral squamous cell carcinoma, however, is known to differ depending on the anatomical site of the primary tumor, such as the tongue, gingiva, mouth floor, and buccal mucosa. In this study, we evaluated the applicability of this morphometric technique to evaluating the risk for cervical lymph node metastasis in oral squamous cell carcinoma.
A digital image system was used to measure the mean nuclear area, mean nuclear perimeter, nuclear circular rate, ratio of nuclear length to width (aspect ratio), and nuclear area coefficient of variation (NACV). Relationships between these parameters and nodal status were evaluated by t-test and logistic regression analysis.
Eighty-eight cases of squamous cell carcinoma (52 of the tongue, 25 of the gingiva, 4 of the buccal mucosa, and 7 of the mouth floor) were included: 46 with positive node classification and 42 with negative node classification. Nuclear area and perimeter were significantly larger in node-positive cases than in node-negative cases; however, there were no significant differences in circular rate, aspect ratio, or NACV. We derived two risk models based on the results of multivariate analysis: Model 1, which identified age and mean nuclear area and Model 2, which identified age and mean nuclear perimeter. It should be noted that primary tumor site was not associated the pN-positive status. There were no significant differences in pathological nodal status by aspect ratio, NACV, or primary tumor site.
Our method of preoperative nuclear morphometry may contribute valuable information to evaluations of the risk for lymph node metastasis in oral squamous cell carcinoma.
PMCID: PMC4280216  PMID: 25549090
10.  Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe 
PLoS ONE  2014;9(12):e115121.
Background and Purpose
To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.
Materials and Methods
DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/+ and p53-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.
The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation.
Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.
PMCID: PMC4274003  PMID: 25531293
11.  Development of Robust Behaviour Recognition for an at-Home Biomonitoring Robot with Assistance of Subject Localization and Enhanced Visual Tracking 
The Scientific World Journal  2014;2014:280207.
Our research is focused on the development of an at-home health care biomonitoring mobile robot for the people in demand. Main task of the robot is to detect and track a designated subject while recognizing his/her activity for analysis and to provide warning in an emergency. In order to push forward the system towards its real application, in this study, we tested the robustness of the robot system with several major environment changes, control parameter changes, and subject variation. First, an improved color tracker was analyzed to find out the limitations and constraints of the robot visual tracking considering the suitable illumination values and tracking distance intervals. Then, regarding subject safety and continuous robot based subject tracking, various control parameters were tested on different layouts in a room. Finally, the main objective of the system is to find out walking activities for different patterns for further analysis. Therefore, we proposed a fast, simple, and person specific new activity recognition model by making full use of localization information, which is robust to partial occlusion. The proposed activity recognition algorithm was tested on different walking patterns with different subjects, and the results showed high recognition accuracy.
PMCID: PMC4283271  PMID: 25587560
12.  Prosocial Behaviors during School Activities among Child Survivors after the 2011 Earthquake and Tsunami in Japan: A Retrospective Observational Study 
PLoS ONE  2014;9(11):e113709.
The 2011 Japan massive tsunami traumatized many children. The aim of this study was to assess changes in strengths and difficulties experienced in home and school by among surviving children after the 2011 tsunami, in comparison with published normal Japanese data.
In November 2012 (20 months after the disaster) and September 2013 (30 months after the disaster), the Strengths and Difficulties Questionnaire (SDQ), a questionnaire on children's strengths and difficulties in home and school activities, were distributed to 12,193 and 11,819 children, respectively. An effective response of children 20 months and 30 month after the disaster was obtained in 10,597 children (86.9%), and 10,812 children (91.4%), respectively. The SDQ scores evaluated by parents and teachers were compared with published normal Japanese SDQ scores.
The SDQ scores (emotional problems, conduct problems, hyperactivity/inattention, peer relationship problems, and total difficulty score) evaluated by parents of children in the 4th to 9th grade who were evaluated after 30 and 20 months were significantly high compared with the published normal data of children without traumatic experiences (all P<0.001). The SDQ scores (prosocial behavior) evaluated by teachers of children in the 4th to 9th grade who were evaluated after 30 and 20 months were significantly low compared with the published normal data of children without traumatic experiences (all P<0.001).
This study showed that the experience of the disaster affected those children with prosocial behaviors towards teachers and friends at school. However, no significant changes (in their prosocial attitude) had been seen at home, where they continued to keep their respect and caring feelings for parents. These results indicate that for accurate diagnosis, clinicians should not only evaluate these children's daily activities at home but also try to objectively assess their daily activities at school.
PMCID: PMC4240583  PMID: 25415450
13.  Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris 
We developed a method for efficient chromosome tagging in Pichia pastoris, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from P. pastoris. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.
PMCID: PMC4237914  PMID: 25398586
Epitope tagging; RNA polymerase; Transcription; Yeast
14.  A Case of Rathke's Cleft Cyst Associated with Transient Central Adrenal Insufficiency and Masked Diabetes Insipidus 
Case Reports in Endocrinology  2014;2014:693294.
A 73-year-old woman admitted to our hospital because of headache, poor appetite, malaise, weight loss, and vomiting was found to have central adrenal insufficiency and thyrotoxicosis due to silent thyroiditis. Polyuria developed after replacement with glucocorticoid (masked diabetes insipidus), which was controlled with nasal administration of desmopressin. Magnetic resonance imaging of the brain showed a large cystic pituitary mass (18 × 18 × 12 mm) extending suprasellarly to the optic chiasm. Transsphenoidal surgery revealed that the pituitary tumor was Rathke's cleft cyst. Following surgery, replacement with neither glucocorticoid nor desmopressin was needed any more. Therefore, it is suggested that Rathke's cleft cyst is responsible for the masked diabetes insipidus and the central insufficiency. Furthermore, it is speculated that thyrotoxicosis with painless thyroiditis might induce changes from subclinical adrenal insufficiency to transiently overt insufficiency.
PMCID: PMC4241302  PMID: 25431697
15.  3D Bioelectronic Interface: Capturing Circulating Tumor Cells onto Conducting Polymer-Based Micro/Nanorod Arrays with Chemical and Topographical Control 
PMCID: PMC4125486  PMID: 24700425
Poly(3,4-ethylenedioxythiophene) (PEDOT); Bioelectronic Interface; Cancer Diagnosis; Circulating Tumor Cells; Nanostructured Materials
16.  Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions 
Cell Death & Disease  2014;5(11):e1517-.
Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.
PMCID: PMC4260733  PMID: 25393473
17.  Arsenite induces premature senescence via p53/p21 pathway as a result of DNA damage in human malignant glioblastoma cells 
BMB Reports  2014;47(10):575-580.
In this study, we investigate whether arsenite-induced DNA damage leads to p53-dependent premature senescence using human glioblastoma cells with p53-wild type (U87MG-neo) and p53 deficient (U87MG-E6). A dose dependent relationship between arsenite and reduced cell growth is demonstrated, as well as induced γH2AX foci formation in both U87MG-neo and U87MG-E6 cells at low concentrations of arsenite. Senescence was induced by arsenite with senescence-associated β-galactosidase staining. Dimethyl- and trimethyl-lysine 9 of histone H3 (H3DMK9 and H3TMK9) foci formation was accompanied by p21 accumulation only in U87MG-neo but not in U87MG-E6 cells. This suggests that arsenite induces premature senescence as a result of DNA damage with heterochromatin forming through a p53/p21 dependent pathway. p21 and p53 siRNA consistently decreased H3TMK9 foci formation in U87M G-neo but not in U87MG-E6 cells after arsenite treatment. Taken together, arsenite reduces cell growth independently of p53 and induces premature senescence via p53/p21-dependent pathway following DNA damage. [BMB Reports 2014; 47(10): 575-580]
PMCID: PMC4261516  PMID: 24499675
Arsenite; Glioma; Heterochromatin formation; Premature senescence; p53
18.  Socioeconomic and Gender Inequalities in Job Dissatisfaction among Japanese Civil Servants: The Roles of Work, Family and Personality Characteristics 
Industrial Health  2014;52(6):498-511.
Abstract: This study examines (1) whether there are employment grade and gender differences in job dissatisfaction and (2) whether work, family, and personality characteristics explain grade and gender differences in job dissatisfaction. The participants were 3,812 civil servants, aged 20–65, working at a local government in Japan. In both males and females, low control, low social support, work-to-family conflict, type A behaviour pattern and negative affectivity were significantly associated with job dissatisfaction. In females, high demands, long work hours and being unmarried were also associated with job dissatisfaction. Among males, in comparison with the highest grade employees, the age-adjusted odds ratio (OR) for job dissatisfaction in the lowest grade employees was 1.90 (95% CI: 1.40–2.59). The grade differences reduced to 1.08 (0.76–1.54) after adjustment for work, family and personality characteristics. Among females, similar grade differences were observed, although the differences were not statistically significant. In comparison with males, the age-adjusted OR in females for job dissatisfaction was 1.32 (1.14–1.52). This gender difference was reduced to 0.95 (0.79–1.14) following adjustment for the other factors. The majority of employees belong to low to middle grades, and female employees have increased. Reducing grade and gender differences in work and family characteristics is needed.
PMCID: PMC4273018  PMID: 25055848
Affect balance; Employment grade; Job satisfaction; Psychosocial stress; Socioeconomic status (SES); The Japanese civil servants study (the JACS study); Type A behaviour; Work-family balance
19.  Breast hemangioma with difficulty in preoperative diagnosis: a case report 
We report a rare case of breast hemangioma found in a 70-year-old Japanese female. Before seeking medical attention, the patient noticed a hard mass in her right breast but denied associated symptoms. A mammography revealed a well-circumscribed, highly dense, lobular nodule located in the middle inter portion of the right breast. To verify this finding, we used ultrasonography which revealed an irregular, iso-echoic nodule measuring 10 mm in the same portion. Based on these findings, we suspected a malignancy and performed a core needle biopsy. Unexpectedly, a histological examination of the biopsy displayed normal vasculature, adipose, and mammary tissues. In order to make an accurate diagnosis, the mass was surgically excised under general anesthesia and sent to pathology. Pathological findings of the mass were positive for breast hemangioma, and the patient has had no recurrence of the disease for the past 24 months.
Electronic supplementary material
The online version of this article (doi:10.1186/1477-7819-12-313) contains supplementary material, which is available to authorized users.
PMCID: PMC4200173  PMID: 25312096
Breast hemangioma; Breast hemangiosarcoma
20.  Chronic obstructive pulmonary disease as a risk factor for lung cancer 
The association between chronic obstructive pulmonary disease (COPD) and lung cancer has long been a subject of intense debate. The high prevalence of COPD in elderly smokers inevitably strengthens their coincidence. In addition to this contingent coincidence, recent studies have revealed a close association between the two diseases that is independent of the smoking history; that is, the existence of COPD is an independent risk factor for the development of lung cancer. Molecular-based evidence has been accumulating as a result of the efforts to explain the underlying mechanisms of this association. These mechanisms may include the following: the retention of airborne carcinogens followed by the activation of oncogenes and the suppression of tumor suppressor genes; the complex molecular mechanism associated with chronic inflammation in the distal airways of patients with COPD; the possible involvement of putative distal airway stem cells; and genetic factors that are common to both COPD and lung cancer. The existence of COPD in patients with lung cancer may potentially affect the process of diagnosis, surgical resection, radiotherapy, chemotherapy, and end-of-life care. The comprehensive management of COPD is extremely important for the appropriate treatment of lung cancer. Surgical resections with the aid of early interventions for COPD are often possible, even for patients with mild-to-moderate COPD. New challenges, such as lung cancer CT screening for individuals at high risk, are now in the process of being implemented. Evaluating the risk of lung cancer in patients with COPD may be warranted in community-based lung cancer screening.
PMCID: PMC4129530  PMID: 25300704
Chronic obstructive pulmonary disease; Airflow limitation; Inflammation; Lung cancer; Carcinogenesis; Cancer screening; Computed tomography screening; Early intervention
21.  Activation of fibroblast-like synoviocytes derived from rheumatoid arthritis via lysophosphatidic acid–lysophosphatidic acid receptor 1 cascade 
Lysophosphatidic acid (LPA) is a bioactive lipid that binds to G protein–coupled receptors (LPA1–6). Recently, we reported that abrogation of LPA receptor 1 (LPA1) ameliorated murine collagen-induced arthritis, probably via inhibition of inflammatory cell migration, Th17 differentiation and osteoclastogenesis. In this study, we examined the importance of the LPA–LPA1 axis in cell proliferation, cytokine/chemokine production and lymphocyte transmigration in fibroblast-like synoviocytes (FLSs) obtained from the synovial tissues of rheumatoid arthritis (RA) patients.
FLSs were prepared from synovial tissues of RA patients. Expression of LPA1–6 was examined by quantitative real-time RT-PCR. Cell surface LPA1 expression was analyzed by flow cytometry. Cell proliferation was analyzed using a cell-counting kit. Production of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), chemokine (C-C motif) ligand 2 (CCL2), metalloproteinase 3 (MMP-3) and chemokine (C-X-C motif) ligand 12 (CXCL12) was measured by enzyme-linked immunosorbent assay. Pseudoemperipolesis was evaluated using a coculture of RA FLSs and T or B cells. Cell motility was examined by scrape motility assay. Expression of adhesion molecules was determined by flow cytometry.
The expression of LPA1 mRNA and cell surface LPA1 was higher in RA FLSs than in FLSs from osteoarthritis tissue. Stimulation with LPA enhanced the proliferation of RA FLSs and the production of IL-6, VEGF, CCL2 and MMP-3 by FLSs, which were suppressed by an LPA1 inhibitor (LA-01). Ki16425, another LPA1 antagonist, also suppressed IL-6 production by LPA-stimulated RA FLSs. However, the production of CXCL12 was not altered by stimulation with LPA. LPA induced the pseudoemperipolesis of T and B cells cocultured with RA FLSs, which was suppressed by LPA1 inhibition. In addition, LPA enhanced the migration of RA FLSs and expression of vascular cell adhesion molecule and intercellular adhesion molecule on RA FLSs, which were also inhibited by an LPA1 antagonist.
Collectively, these results indicate that LPA–LPA1 signaling contributes to the activation of RA FLSs.
PMCID: PMC4203966  PMID: 25273676
22.  Expression of GLUT-1 and GLUT-3 in Xanthogranulomatous Cholecystitis Induced a Positive Result on 18F-FDG PET: Report of a Case 
International Surgery  2013;98(4):372-378.
Although several reports have revealed that fluorine-18 fluorodeoxyglucose positron emission tomography (18F-FDG PET) is useful for differentiating between benign and malignant lesions in the gallbladder, the positive results of 18F-FDG PET are not specific for malignancy because 18F-FDG is also accumulated in inflammatory lesions. It is known that the most important pathway for 18F-FDG to enter the cell body is mediated by the facilitative glucose transporter-1 (GLUT-1) through GLUT-3. We herein present a case of xanthogranulomatous cholecystitis (XGC) with a positive result on 18F-FDG PET. In this case, GLUT-1 and GLUT-3 were both positively expressed in inflammatory cells at the gallbladder wall of XGC and this is the first report to reveal GLUT expression in XGC. This report reveals that surgeons should carefully consider the appropriate treatment of gallbladder tumor, even with a positive result on 18F-FDG PET.
PMCID: PMC3829066  PMID: 24229026
Xanthogranulomatous cholecystitis; Fluorine-18 fluorodeoxyglucose positron emission tomography; GLUTs
23.  Engineering of human hepatic tissue with functional vascular networks 
Organogenesis  2014;10(2):260-267.
Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.
PMCID: PMC4154961  PMID: 24451152
fetal liver cells; liver; mesenchymal stem cell; tissue engineering; vascular network
24.  A Macroscopic Reaction: Direct Covalent Bond Formation between Materials Using a Suzuki-Miyaura Cross-Coupling Reaction 
Scientific Reports  2014;4:6348.
Cross-coupling reactions are important to form C–C covalent bonds using metal catalysts. Although many different cross-coupling reactions have been developed and applied to synthesize complex molecules or polymers (macromolecules), if cross-coupling reactions are realized in the macroscopic real world, the scope of materials should be dramatically broadened. Here, Suzuki-Miyaura coupling reactions are realized between macroscopic objects. When acrylamide gel modified with an iodophenyl group (I-gel) reacts with a gel possessing a phenylboronic group (PB-gel) using a palladium catalyst, the gels bond to form a single object. This concept can also be adapted for bonding between soft and hard materials. I-gel or PB-gel selectively bonds to the glass substrates whose surfaces are modified with an electrophile or nucleophile, respectively.
PMCID: PMC4166947  PMID: 25231557
25.  The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90 
FEBS Open Bio  2014;4:796-803.
•We showed the direct interaction of AhR and HSP90 using purified protein.•The ligand 17-DMAG induces a dissociation of HSP90 from AhR.•The AhR–HSP90 complex is not affected by the timing of β-naphthoflavone binding to AhR.•The AhR–HSP90 complex was translocated to the nucleus after treatment with β-naphthoflavone.
The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or β-naphthoflavone (β-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to β-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90.
We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with β-NF.
PMCID: PMC4208086  PMID: 25349783
17-DMAG, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, dimethylsulfoxide; 3-MC, 3-methylcholanthrene; AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; bHLH, basic helix-loop-helix; CYP1A1, cytochrome P450 1A1; DAPI, 4′,6-diamidino-2-phenylindole; DEPC, dihydrochloride, diethylpyrocarbonated; GST, glutathione, glutathione S-transferase; HSP90, 90-kDa of heat shock protein; IPTG, isopropyl-1-thio-β-d-galactopyranoside; IPTG, isopropyl-1-thio-β-d-galactopyranoside; NLS, nuclear localization signal; PAS, per-arnt-sim; PLA, proximity ligation assay; RT-PCR, reverse transcription-polymerase chain reaction; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XAP2, hepatitis B virus X-associated protein; XRE, xenobiotic responsible element; β-NF, β-naphthoflavone; Aryl hydrocarbon receptor; AhR; Dioxin receptor; Molecular chaperone; HSP90

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