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author:("harasawa, S.")
1.  Orexin-1 receptor mediates the increased food and water intake induced by intracerebroventricular injection of stable somatostatin pan-agonist, ODT8-SST in rats 
Neuroscience letters  2014;576:88-92.
Intracerebroventricular (icv) injection of the stable somatostatin pan-agonist, ODT8-SST induces a somatostatin 2 receptor (sst2) mediated robust feeding response that involves neuropeptide Y and opioid systems in rats. We investigated whether the orexigenic system driven by orexin also plays a role. Food and water intake after icv injection was measured concomitantly in non-fasted and non-water deprived rats during the light phase. In vehicle treated rats (100% DMSO, icv), ODT8-SST (1 μg/rat, icv) significantly increased the 2-h food and water intake compared to icv vehicle plus saline (5.1 ± 1.0 g vs. 1.2 ± 0.4 g and 11.3 ± 1.9 mL vs. 2.5 ± 1.2 mL, respectively). The orexin-1 receptor antagonist, SB-334867 (16 μg/rat, icv) completely inhibited the 2-h food and water intake induced by icv ODT8-SST. In contrast, the icv pretreatment with the selective somatostatin sst2 antagonist, S-406-028, established to block the orexigenic effect of icv ODT8-SST, did not modify the increased food and water intake induced by icv orexin-A (10.7 μg/rat). These data indicate that orexin-1 receptor signaling system is part of the brain neurocircuitry contributing to the orexigenic and dipsogenic responses induced by icv ODT8-SST and that orexin-A stimulates food intake independently from brain sst2 activation.
doi:10.1016/j.neulet.2014.05.063
PMCID: PMC4096604  PMID: 24915296
food intake; orexin-A; ODT8-SST; SB-334867; somatostatin 2 receptor; water intake
2.  Role of NLRP3 Inflammasomes for Rhabdomyolysis-induced Acute Kidney Injury 
Scientific Reports  2015;5:10901.
Rhabdomyolysis is one of the main causes of community-acquired acute kidney injury (AKI). Although inflammation is involved in the pathogenesis of rhabdomyolysis-induced AKI (RIAKI), little is known about the mechanism that triggers inflammation during RIAKI. Recent evidence has indicated that sterile inflammation triggered by tissue injury can be mediated through multiprotein complexes called the inflammasomes. Therefore, we investigated the role of NLRP3 inflammasomes in the pathogenesis of RIAKI using a glycerol-induced murine rhabdomyolysis model. Inflammasome-related molecules were upregulated in the kidney of RIAKI. Renal tubular injury and dysfunction preceded leukocyte infiltration into the kidney during the early phase of RIAKI, and they were markedly attenuated in mice deficient in NLRP3, ASC, caspase-1, and interleukin (IL)-1β compared with those in wild-type mice. No difference in leukocyte infiltration was observed between wild-type and NLRP3-deficient mice. Furthermore, NLRP3 deficiency strikingly suppressed the expression of renal injury markers and inflammatory cytokines and apoptosis of renal tubular cells. These results demonstrated that NLRP3 inflammasomes contribute to inflammation, apoptosis, and tissue injury during the early phase of RIAKI and provide new insights into the mechanism underlying the pathogenesis of RIAKI.
doi:10.1038/srep10901
PMCID: PMC4456665  PMID: 26045078
3.  Clinical Trial of Prophylactic Extended-Field Carbon-Ion Radiotherapy for Locally Advanced Uterine Cervical Cancer (Protocol 0508) 
PLoS ONE  2015;10(5):e0127587.
To evaluate the efficacy and the toxicity of prophylactic extended-field carbon-ion radiotherapy (C-ion RT, Protocol 0508) for locally advanced squamous cell carcinoma of the uterine cervix in phase I / II clinical trial. Between May 2006 and January 2012, 26 patients of Protocol 0508 were treated with C-ion RT. The numbers of patients with stage IIB, IIIB, and IVA disease were 13, 11, and 2, respectively. Twenty patients had pelvic lymph node metastases. Median tumor size was 6.1 cm (range, 4.0–10.0 cm). The treatment consisted of extended-field irradiation of 39.0 gray equivalents (GyE) in 13 fractions, and additional 15.0 GyE in 5 fractions was given to the gross tumor volume (GTV) and surrounding tissues. With regard to local boost, 18.0 GyE in 2 fractions was given to GTV only. Total dose to the cervical tumor was 72.0 GyE over 20 fractions. The median follow-up period was 37 months. Twenty-one patients had grade 1 or 2 acute gastrointestinal toxicity, but all patients completed the treatment on schedule. There were no grade 3 or higher late complications, with 8 patients having grade 1 or 2 toxicities, 1 had grade 2 gastrointestinal toxicity and 2 had grade 2 genitourinary toxicity. Four patients (15.4%) developed local recurrence, and 8 patients (30.8%) had distant metastases. The 2-year local control rate, progression-free survival rate and overall survival rate were 83.6%, 61.5% and 73.1%, respectively. There were no severe acute or late complications in this trial. Prophylactic extended-field C-ion RT for locally advanced squamous cell carcinoma of the uterine cervix was a safe treatment. Although the number of patients in this study was small, the results support further investigations to confirm the therapeutic efficacy and to avoid or reduce toxicity.
Trial Registration
UMIN-CTR UMIN000016169
doi:10.1371/journal.pone.0127587
PMCID: PMC4439043  PMID: 25993047
4.  Cripto-1 as a novel therapeutic target for triple negative breast cancer 
Oncotarget  2014;6(14):11910-11929.
Triple-negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes and no current standard therapy. Here, we performed an in-depth molecular analysis of a mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resembled the triple-negative breast cancer (TNBC) both phenotypically and molecularly. Morphologically, primary tumors presented both epithelial and spindle-like cells but displayed only adenocarcinoma-like features in lung parenchyma. The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively. Cripto-1, an embryonic stem cell marker, was present in spindle-like areas and its promoter showed activity in primary tumors. Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis. Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.
PMCID: PMC4494913  PMID: 26059540
cripto-1; notch4; epithelial-mesenchymal plasticity; mouse model; triple-negative breast cancer
5.  Diverse Kir Expression Contributes to Distinct Bimodal Distribution of Resting Potentials and Vasotone Responses of Arterioles 
PLoS ONE  2015;10(5):e0125266.
The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba2+ 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K+, while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K+ typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba2+-sensitive inward rectifier K+ (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir-based regenerative shifts between two RP states could form a critical mechanism for conduction/spread of vasomotion along the arteriole axis.
doi:10.1371/journal.pone.0125266
PMCID: PMC4418701  PMID: 25938437
6.  Current status and outcomes of patients developing PSA recurrence after prostatectomy who were treated with salvage radiotherapy: a JROSG surveillance study 
Journal of Radiation Research  2015;56(4):750-756.
The conditions and outcomes of Japanese patients with prostate cancer who developed PSA failure after radical prostatectomy (RP), and who were treated via salvage radiotherapy (S-RT), were surveyed. Clinical data on S-RT were gathered in questionnaires completed by facilities participating in the Japanese Radiation Oncology Study Group. S-RT was defined as external-beam radiotherapy delivered to the prostate beds of patients with prostate cancer who had eventually developed PSA failure, although their PSA values had at one stage attained levels <0.2 ng/ml following RP. Hormonal therapy was combined with S-RT in ∼40% of cases. Outcomes were evaluated in 186 cases treated via S-RT alone. The nadir PSA level after RP, and the level upon initiation of S-RT, were 0.0135 ng/ml and 0.292 ng/ml, respectively. The median period between RP and S-RT was 18.6 months. The median follow-up period was 58 months. The 5-year PSA recurrence–free survival (PRFS) and clinical failure–free survival (CFFS) rates were 50.1% (95% CI: 42.8–57.9%) and 90.1% (95% CI: 86.4–95.7%), respectively. PRFS was significantly superior in patients with PSA values ≤0.3 ng/ml upon initiation of S-RT than in those with PSA values >0.3 ng/ml (57.5% vs 40.5%, P = 0.027). In Japan, hormonal therapy is combined with S-RT in ∼40% of cases. The 5-year PRFS and CFFS rates of cases treated via S-RT alone were 50.1% and 90.1%, respectively. A PSA value of 0.3 ng/ml served as a significant cut-off for prediction of PRFS.
doi:10.1093/jrr/rrv027
PMCID: PMC4497400  PMID: 25910489
salvage radiotherapy; prostate cancer; PSA recurrence; radical prostatectomy
7.  Oligomerized CARD16 promotes caspase-1 assembly and IL-1β processing 
FEBS Open Bio  2015;5:348-356.
Highlights
•CARD16 is the most abundant CARD-only protein in hematopoietic cells.•Unlike CARD17, CARD16 oligomerizes to form a filament-like structure.•The filament-like structure formed by CARD16 promotes caspase 1 (CASP1) assembly.•CASP1-dependent IL-1β processing is enhanced by CARD16.•CARD16 colocalizes in ASC-speck by interacting with ASC.
Increasing evidence indicates that caspase recruitment domain (CARD)-mediated caspase-1 (CASP1) assembly is an essential process for its activation and subsequent interleukin (IL)-1β release, leading to the initiation of inflammation. Both CARD16 and CARD17 were previously reported as inhibitory homologs of CASP1; however, their molecular function remains unclear. Here, we identified that oligomerization activity allows CARD16 to function as a CASP1 activator. We investigated the molecular characteristics of CARD16 and CARD17 in transiently transfected HeLa cells. Although both CARD16 and CARD17 interacted with CASP1CARD, only CARD16 formed a homo-oligomer. Oligomerized CARD16 formed a filament-like structure with CASP1CARD and a speck with apoptosis-associated speck-like protein containing a CARD. A filament-like structure formed by CARD16 promoted CASP1 filament assembly and IL-1β release. In contrast, CARD17 did not form a homo-oligomer or filaments and inhibited CASP1-dependent IL-1β release. Mutated CARD16D27G, mimicking the CARD17 amino acid sequence, formed a homo-oligomer but failed to form a filament-like structure. Consequently, CARD16D27G weakly promoted CASP1 filament assembly and subsequent IL-1β release. These results suggest that oligomerized CARD16 promotes CARD-mediated molecular assembly and CASP1 activation.
doi:10.1016/j.fob.2015.04.011
PMCID: PMC4420773  PMID: 25973362
ANOVA, analysis of variance; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; Bcl10, B-cell lymphoma/leukemia 10; BS3, bis(sulfosccinimidyl)suberate; CARD, caspase recruitment domain; CARMA1, CARD-membrane-associated guanylate kinase 1; COPs, CARD-only proteins; CASP1, caspase-1; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; IL, interleukin; LPS, lipopolysaccharide; LRRs, leucine-rich repeats; NLRs, nucleotide-binding domain leucine-rich repeat containing receptors; NF-κB, nuclear factor kappa beta; PYD, pyrin domain; Caspase; Cytokine; Inflammation; Interleukin
8.  An FDG-PET/CT-Positive Lesion Mimicking Local Recurrence of Colon Cancer 5 Years after Radical Colectomy 
Patient: Female, 75
Final Diagnosis: False positive findings
Symptoms: —
Medication: —
Clinical Procedure: —
Specialty: Surgery
Objective:
Mistake in diagnosis
Background:
Radical resection of colorectal cancer yields satisfactory results. Even if the cancer recurs, long-term survival is expected through further surgical resection of the recurrent disease. For early detection of recurrent lesions, we routinely perform periodic blood tests and imaging studies, in which 18F-fluorodeoxyglucose-glucose positron emission tomography (FDG-PET) plays an important role, for lesion differentiation. We encountered a case of a benign lesion, which had been clinically diagnosed as recurrence of resected colon cancer by FDG-PET/computed tomography (CT).
Case Report:
A 69-year-old woman underwent radical resection of stage II sigmoid colon cancer. Five years after the operation, local recurrence was suspected on the basis of follow-up CT examination findings. Since the standardized uptake value (SUV) on FDG-PET/CT was 13.3, we diagnosed the lesion as a postoperative local recurrence and performed surgical resection of the lesion. The lesion was conclusively diagnosed as benign fatty tissue, including a fibrovascular component, by histopathological examination.
Conclusions:
FDG-PET is a very useful technique for differentiating benign from malignant disease. In colorectal cancer, FDGPET not only enables the differentiation of malignancy in the primary tumor, but also the confirmation of metastasis and postoperative recurrence. However, even if the SUV is high, as in the presented case, the lesion may eventually be diagnosed as benign. Therefore, further advances in the PET technique are expected along with the development of more useful modalities.
doi:10.12659/AJCR.891129
PMCID: PMC4370278  PMID: 25761604
Colorectal Neoplasms; Positron-Emission Tomography; Neoplasm Recurrence; Local
9.  Culture, inequality, and health: evidence from the MIDUS and MIDJA comparison 
Culture and Brain  2015;3(1):1-20.
This article seeks to forge scientific connections between three overarching themes (culture, inequality, health). Although the influence of cultural context on human experience has gained notable research prominence, it has rarely embraced another large arena of science focused on the influence social hierarchies have on how well and how long people live. That literature is increasingly focused psychosocial factors, working interactively with biological and brain-based mechanisms, to account for why those with low socioeconomic standing have poorer health. Our central question is whether and how these processes might vary by cultural context. We draw on emerging findings from two parallel studies, Midlife in the U.S. and Midlife in Japan, to illustrate the cultural specificity evident in how psychosocial and neurobiological factors are linked with each other as well as how position in social hierarchies matters for psychological experience and biology. We conclude with suggestions for future multidisciplinary research seeking to understand how social hierarchies matter for people’s health, albeit in ways that may possibly differ across cultural contexts.
doi:10.1007/s40167-015-0025-0
PMCID: PMC4342505  PMID: 25750852
Culture; Inequality; Health; MIDUS; MIDJA
10.  Difference in distant failure site between locally advanced squamous cell carcinoma and adenocarcinoma of the uterine cervix after C-ion RT 
Journal of Radiation Research  2015;56(3):523-528.
We investigated the first site of distant failure after carbon ion radiotherapy (C-ion RT) for locally advanced cervical cancer in three clinical trials. A total of 91 cases were enrolled in the three trials (Protocol 9702, 9704 and 9902). Histologically, 36 cases had squamous cell carcinoma (SqCC) and 55 cases had adenocarcinoma (AC), including 13 with adenosquamous cell carcinoma. The number of cases with Stage IIB, IIIB and IVA disease was 21, 59 and 11, respectively. Of the 91 cases, 42 had positive pelvic lymph nodes (PLNs). The median tumor size was 6.0 cm (range, 3.0–12.0 cm). The median follow-up duration for all cases was 40 months (range, 7–181 months). A total of 40 cases developed distant failure as the first site of failure: 13 of 36 (36.1%) SqCC cases had distant failure, with 9 of them with para-aortic lymph node (PALN) failure; 27 of 55 (44.0%) AC cases had distant failure, and 23 of them had distant failure excluding PALN metastasis. Distant failure rates of SqCC cases who had positive and negative PLNs before C-ion RT were 61.1% and 11.1%, respectively (P = 0.0045). Those of AC cases were 54.2% and 45.2%, respectively (P = 0.507). In conclusion, there were high rates of distant failure after C-ion RT in AC cases regardless of PLN status, and there were high rates of distant failure after C-ion RT, especially PALN failure, in SqCC cases with positive PLNs.
doi:10.1093/jrr/rru117
PMCID: PMC4426912  PMID: 25589503
carbon-ion radiotherapy; uterine cervical cancer; distant failure; adenocarcinoma of the uterine cervix; para-aortic lymph node metastasis
11.  Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. ‘Monreal Rosso’ and ‘Nules’ 
Microspore embryogenesis is a method of achieving complete homozygosity from plants. It is particularly useful for woody species, like Citrus, characterized by long juvenility, a high degree of heterozygosity and often self-incompatibility. Anther culture is currently the method of choice for microspore embryogenesis in many crops. However, isolated microspore culture is a better way to investigate the processes at the cellular, physiological, biochemical, and molecular levels as it avoids the influence of somatic anther tissue. To exploit the potential of this technique, it is important to separate the key factors affecting the process and, among them, culture medium composition and particularly the plant growth regulators and their concentration, as they can greatly enhance regeneration efficiency. To our knowledge, the ability of meta-Topolin, a naturally occurring aromatic cytokinin, to induce gametic embryogenesis in isolated microspores of Citrus has never been investigated. In this study, the effect of two concentrations of meta-Topolin instead of benzyladenine or zeatin in the culture medium was investigated in isolated microspore culture of two genotypes of Citrus. After 11 months of isolated microspore culture, for both genotypes and for all the four tested media, the microspore reprogramming and their sporophytic development was observed by the presence of multinucleated calli and microspore-derived embryos at different stages. Microsatellite analysis of parental and embryo samples was performed to determine the embryo alleles constitution of early embryos produced in all tested media, confirming their origin from microspores. To our knowledge, this is the first successful report of Citrus microspore embryogenesis with isolated microspore culture in Citrus, and in particular in Citrus clementina Hort. ex Tan, cvs. ‘Monreal Rosso’ and ‘Nules.’
doi:10.3389/fpls.2015.00413
PMCID: PMC4463929  PMID: 26124764
citrus breeding; gametic embryogenesis; homozygosity; isolated microspore culture; meta-Topolin
12.  Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages 
PLoS ONE  2014;9(12):e113974.
Background
Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages.
Methods and Results
IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody.
Conclusions
Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.
doi:10.1371/journal.pone.0113974
PMCID: PMC4259327  PMID: 25486008
13.  Negative Emotions Predict Elevated Interleukin-6 in the United States but not in Japan 
Brain, behavior, and immunity  2013;34:10.1016/j.bbi.2013.07.173.
Previous studies conducted in Western cultures have shown that negative emotions predict higher levels of pro-inflammatory biomarkers, specifically interleukin-6 (IL-6). This link between negative emotions and IL-6 may be specific to Western cultures where negative emotions are perceived to be problematic and thus may not extend to Eastern cultures where negative emotions are seen as acceptable and normal. Using samples of 1044 American and 382 Japanese middle-aged and older adults, we investigated whether the relationship between negative emotions and IL-6 varies by cultural context. Negative emotions predicted higher IL-6 among American adults, whereas no association was evident among Japanese adults. Furthermore, the interaction between culture and negative emotions remained even after controlling for demographic variables, psychological factors (positive emotions, neuroticism, extraversion), health behaviors (smoking status, alcohol consumption), and health status (chronic conditions, BMI). These findings highlight the role of cultural context in shaping how negative emotions affect inflammatory physiology and underscore the importance of cultural ideas and practices relevant to negative emotions for understanding of the interplay between psychology, physiology, and health.
doi:10.1016/j.bbi.2013.07.173
PMCID: PMC3826918  PMID: 23911591
culture; negative emotion; inflammation; Interleukin-6
14.  Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides 
PLoS ONE  2014;9(9):e108941.
Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2+/− mice, but not in Sglt2−/− mice. However, serum GTTR levels were elevated in Sglt2−/− mice compared to Sglt2+/− mice, and in phlorizin-treated Sglt2+/− mice compared to vehicle-treated Sglt2+/− mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity.
doi:10.1371/journal.pone.0108941
PMCID: PMC4182564  PMID: 25268124
15.  Deficiency of Transcription Factor Brn4 Disrupts Cochlear Gap Junction Plaques in a Model of DFN3 Non-Syndromic Deafness 
PLoS ONE  2014;9(9):e108216.
Brn4, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome–linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K+ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.
doi:10.1371/journal.pone.0108216
PMCID: PMC4178122  PMID: 25259580
16.  The DNA Repair Enzyme Apurinic/Apyrimidinic Endonuclease (Apex Nuclease) 2 Has the Potential to Protect against Down-Regulation of Chondrocyte Activity in Osteoarthritis 
Apurinic/apyrimidinic endonuclease 2 (Apex 2) plays a critical role in DNA repair caused by oxidative damage in a variety of human somatic cells. We speculated that chondrocyte Apex 2 may protect against the catabolic process of articular cartilage in osteoarthritis (OA). Higher levels of Apex 2 expression were histologically observed in severely compared with mildly degenerated OA cartilage from STR/OrtCrlj mice, an experimental model which spontaneously develops OA. The immunopositivity of Apex 2 was significantly correlated with the degree of cartilage degeneration. Moreover, the OA-related catabolic factor interleukin-1β induced the expression of Apex 2 in chondrocytes, while Apex 2 silencing using small interfering RNA reduced chondrocyte activity in vitro. The expression of Apex 2 in chondrocytes therefore appears to be associated with the degeneration of articular cartilage and could be induced by an OA-related catabolic factor to protect against the catabolic process of articular cartilage. Our findings suggest that Apex 2 may have the potential to prevent the catabolic stress-mediated down-regulation of chondrocyte activity in OA.
doi:10.3390/ijms150914921
PMCID: PMC4200784  PMID: 25158232
osteoarthritis; DNA repair enzyme; oxidative stress; chondrocytes; Apex 2 (apurinic/apyrimidinic endonuclease 2)
17.  Regulation of human Cripto-1 expression by nuclear receptors and DNA promoter methylation in human embryonal and breast cancer cells 
Journal of cellular physiology  2013;228(6):1174-1188.
Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1 and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.
doi:10.1002/jcp.24271
PMCID: PMC3573215  PMID: 23129342
Cripto-1; GCNF; LRH-1; DNA methylation
18.  Bumetanide hyperpolarizes Madin-Darby canine kidney cells and enhances cellular gentamicin uptake via elevating cytosolic Ca2+ thus facilitating intermediate conductance Ca2+-activated potassium channels 
Cell biochemistry and biophysics  2013;65(3):381-398.
Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby Canine kidney (MDCK) cells. We found that bumetanide and furosemide concentration-dependently enhanced cytoplasmic GTTR fluorescence by ~60%. This enhancement was suppressed by La3+, a non-selective cation channel (NSCC) blocker, and by K+ channel blockers Ba2+ and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl− channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (Vr) ~−80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba2+ or clotrimazole, and absent in elevated [Ca2+]i, but not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca2+. Ba2+ and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current Vr near −85 mV, and reduced GTTR uptake by ~20%. La3+ alone hyperpolarized the cells by ~−14 mV, reduced the I/V slope with a net current Vr near −10 mV, and inhibited GTTR uptake by ~50%. In the presence of La3+, bumetanide caused negligible potential or I/V change. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating the intracellular Ca2+ and thus a facilitation of the intermediate conductance Ca2+-activated K+ channels.
doi:10.1007/s12013-012-9442-2
PMCID: PMC3584200  PMID: 23109177
aminoglycoside; ototoxicity; loop diuretics; membrane potential; ion current; chloride channels; cytosolic calcium
19.  Japanese consensus guidelines for pediatric nuclear medicine 
Annals of Nuclear Medicine  2014;28(5):498-503.
The Japanese Society of Nuclear Medicine has recently published the consensus guidelines for pediatric nuclear medicine. This article is the English version of the guidelines. Part 1 proposes the dose optimization in pediatric nuclear medicine studies. Part 2 comprehensively discusses imaging techniques for the appropriate conduct of pediatric nuclear medicine procedures, considering the characteristics of imaging in children.
doi:10.1007/s12149-014-0826-9
PMCID: PMC4061477  PMID: 24647992
20.  Burmoniscus kitadaitoensis Nunomura, 2009 (Crustacea, Isopoda, Oniscidea) from southern Japan, a junior synonym of B. meeusei (Holthuis, 1947) 
ZooKeys  2014;21-28.
Re-examination of the holotype of Burmoniscus kitadaitoensis Nunomura, 2009 from Kitadaitojima Island, southern Japan reveals that this species is a junior synonym of B. meeusei (Holthuis, 1947). Partial regions of mitochondrial COI, 12S and 16S rRNA genes, and nuclear 18S and 28S rRNA genes were detected for species identification in the future.
doi:10.3897/zookeys.386.6727
PMCID: PMC3970063  PMID: 24693213
Kitadaitojima Island; mitochondrial DNA; nuclear DNA; Philosciidae; terrestrial isopods
21.  Assembly of the cochlear gap junction macromolecular complex requires connexin 26 
The Journal of Clinical Investigation  2014;124(4):1598-1607.
Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.
doi:10.1172/JCI67621
PMCID: PMC3973107  PMID: 24590285
22.  Clinical trial of carbon ion radiotherapy for gynecological melanoma 
Journal of Radiation Research  2014;55(2):343-350.
Carbon ion radiotherapy (C-ion RT) is an advanced modality for treating malignant melanoma. After we treated our first case of gynecological melanoma using C-ion RT in November 2004, we decided to conduct a clinical trial to evaluate its usefulness for the treatment of gynecological melanoma. The eligibility criteria for enrollment in this study were histologically proven malignant melanoma of the gynecological regions with lymph node metastasis remaining in the inguinal and pelvic regions. The small pelvic space, including the GTV and the metastatic lymph node, was irradiated with up to a total dose of 36 GyE followed by a GTV boost of up to a total dose of 57.6 GyE or 64 GyE in 16 fractions. A series of 23 patients were treated between November 2004 and October 2012. Patient age ranged from 51–80 with a median of 71. Of the tumor sites, 14 were located in the vagina, 6 in the vulva, and 3 in the cervix uteri. Of the 23 patients, 22 were irradiated with up to a total dose of 57.6 GyE, and 1 patient was irradiated with up to a total dose of 64 GyE. Chemotherapy and interferon-β were also used to treat 11 of the patients. Acute and late toxicities of Grade 3 or higher were observed in 1 patient treated with concurrent interferon-β. The median follow-up time was 17 months (range, 6–53 months). There was recurrence in 14 patients, and the 3-year local control and overall survival rates were 49.9% and 53.0%, respectively. C-ion RT may become a non-invasive treatment option for gynecological melanoma.
doi:10.1093/jrr/rrt120
PMCID: PMC3951082  PMID: 24536019
carbon ion radiotherapy; gynecological melanoma
23.  Dominant negative connexin26 mutation R75W causing severe hearing loss influences normal programmed cell death in postnatal organ of Corti 
BMC Genetics  2014;15:1.
Background
The greater epithelial ridge (GER) is a developmental structure in the maturation of the organ of Corti. Situated near the inner hair cells of neonatal mice, the GER undergoes a wave of apoptosis after postnatal day 8 (P8). We evaluated the GER from P8 to P12 in transgenic mice that carry the R75W + mutation, a dominant-negative mutation of human gap junction protein, beta 2, 26 kDa (GJB2) (also known as connexin 26 or CX26). Cx26 facilitate intercellular communication within the mammalian auditory organ.
Results
In both non-transgenic (non-Tg) and R75W + mice, some GER cells exhibited apoptotic characteristics at P8. In the GER of non-Tg mice, both the total number of cells and the number of apoptotic cells decreased from P8 to P12. In contrast, apoptotic cells were still clearly evident in the GER of R75W + mice at P12. In R75W + mice, therefore, apoptosis in the GER persisted until a later stage of cochlear development. In addition, the GER of R75W + mice exhibited morphological signs of retention, which may have resulted from diminished levels of apoptosis and/or promotion of cell proliferation during embryogenesis and early postnatal stages of development.
Conclusions
Here we demonstrate that Cx26 dysfunction is associated with delayed apoptosis of GER cells and GER retention. This is the first demonstration that Cx26 may regulate cell proliferation and apoptosis during development of the cochlea.
doi:10.1186/1471-2156-15-1
PMCID: PMC3893426  PMID: 24387126
Apoptosis; Hereditary hearing loss; Gjb2; Greater epithelial ridge; Mouse; Organ of corti
24.  Cripto-1 enhances the canonical Wnt/β-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors 
Cellular signalling  2012;25(1):178-189.
Cripto-1 is implicated in multiple cellular events, including cell proliferation, motility and angiogenesis, through the activation of an intricate network of signaling pathways. A crosstalk between Cripto-1 and the canonical Wnt/β-catenin signaling pathway has been previously described. In fact, Cripto-1 is a downstream target gene of the canonical Wnt/β-catenin signaling pathway in the embryo and in colon cancer cells and T-cell factor (Tcf)/lymphoid enhancer factor binding sites have been identified in the promoter and the first intronic region of the mouse and human Cripto-1 genes. We now demonstrate that Cripto-1 modulates signaling through the canonical Wnt/β-catenin/Tcf pathway by binding to the Wnt co-receptors low-density lipoprotein receptor-related protein (LRP) 5 and LRP6, which facilitates Wnt3a binding to LRP5 and LRP6. Cripto-1 functionally enhances Wnt3a signaling through cytoplasmic stabilization of β-catenin and elevated β-catenin/Tcf transcriptional activation. Conversely, Wnt3a further increases Cripto-1 stimulation of migration, invasion and colony formation in soft agar of HC11 mouse mammary epithelial cells, indicating that Cripto-1 and the canonical Wnt/β-catenin signaling co-operate in regulating motility and in vitro transformation of mammary epithelial cells.
doi:10.1016/j.cellsig.2012.09.024
PMCID: PMC3508164  PMID: 23022962
Cripto-1; Wnt signaling; β-catenin; LRP5; LRP6
25.  Social Status and Anger Expression: The Cultural Moderation Hypothesis 
Emotion (Washington, D.C.)  2013;13(6):10.1037/a0034273.
Individuals with lower social status have been reported to express more anger, but this evidence comes mostly from Western cultures. Here, we used representative samples of American and Japanese adults and tested the hypothesis that the association between social status and anger expression depends on whether anger serves primarily to vent frustration, as in the United States, or to display authority, as in Japan. Consistent with the assumption that lower social standing is associated with greater frustration stemming from life adversities and blocked goals, Americans with lower social status expressed more anger, with the relationship mediated by the extent of frustration. In contrast, consistent with the assumption that higher social standing affords a privilege to display anger, Japanese with higher social status expressed more anger, with the relationship mediated by decision-making authority. As expected, anger expression was predicted by subjective social status among Americans and by objective social status among Japanese. Implications for the dynamic construction of anger and anger expression are discussed.
doi:10.1037/a0034273
PMCID: PMC3859704  PMID: 24098926
anger expression; culture; social status; independence and interdependence

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