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author:("harasawa, S.")
1.  Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages 
PLoS ONE  2014;9(12):e113974.
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.
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.
PMCID: PMC4259327  PMID: 25486008
2.  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.
PMCID: PMC3826918  PMID: 23911591
culture; negative emotion; inflammation; Interleukin-6
3.  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.
PMCID: PMC4182564  PMID: 25268124
4.  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.
PMCID: PMC4178122  PMID: 25259580
5.  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.
PMCID: PMC4200784  PMID: 25158232
osteoarthritis; DNA repair enzyme; oxidative stress; chondrocytes; Apex 2 (apurinic/apyrimidinic endonuclease 2)
6.  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.
PMCID: PMC3573215  PMID: 23129342
Cripto-1; GCNF; LRH-1; DNA methylation
7.  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.
PMCID: PMC3584200  PMID: 23109177
aminoglycoside; ototoxicity; loop diuretics; membrane potential; ion current; chloride channels; cytosolic calcium
8.  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.
PMCID: PMC4061477  PMID: 24647992
9.  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.
PMCID: PMC3970063  PMID: 24693213
Kitadaitojima Island; mitochondrial DNA; nuclear DNA; Philosciidae; terrestrial isopods
10.  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.
PMCID: PMC3973107  PMID: 24590285
11.  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.
PMCID: PMC3951082  PMID: 24536019
carbon ion radiotherapy; gynecological melanoma
12.  Dominant negative connexin26 mutation R75W causing severe hearing loss influences normal programmed cell death in postnatal organ of Corti 
BMC Genetics  2014;15:1.
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.
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.
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.
PMCID: PMC3893426  PMID: 24387126
Apoptosis; Hereditary hearing loss; Gjb2; Greater epithelial ridge; Mouse; Organ of corti
13.  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.
PMCID: PMC3508164  PMID: 23022962
Cripto-1; Wnt signaling; β-catenin; LRP5; LRP6
14.  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.
PMCID: PMC3859704  PMID: 24098926
anger expression; culture; social status; independence and interdependence
15.  Long-term survival after sequential chemotherapy and surgery for advanced gastric cancer☆ 
We experienced a case with long relapse-free survival after successful treatment of chemotherapy and surgery to advanced gastric cancer.
A 56-year-old man was examined because of rapid weight loss and was diagnosed as having far-advanced gastric cancer with portal vein tumor thrombus (PVTT) and liver, lymph node and peritoneal metastases. Immediately after beginning chemotherapy, gastric obstruction due to gastric cancer was discovered. Therefore gastrojejunostomy, a bypass operation, was performed, and this was followed by the first course chemotherapy with S-1 and cisplatin. After 4 courses of this regimen were completed, PVTT and the peritoneal metastasis could no longer be confirmed, and new lesion had not appeared; therefore, the patient underwent a radical operation with distal gastrectomy, lymph node dissection and partial hepatectomy. After the operation, he received second-line chemotherapy with S-1 and paclitaxel for 1 year. He has been in good health without any signs of recurrence for 3 years and 8 months after the radical operation.
Although complete recovery from far-advanced gastric cancer is rarely expected, this case demonstrates that long-term survival is achievable with carefully considered treatment plans.
PMCID: PMC3825979  PMID: 24070832
Gastric cancer; Portal vein tumor thrombus; Liver metastasis; Peritoneal dissemination; Gastrojejunostomy
16.  Impact of boost irradiation on pelvic lymph node control in patients with cervical cancer 
Journal of Radiation Research  2013;55(1):139-145.
Radiation therapy (RT) for metastatic pelvic lymph nodes (PLNs) is not well established in cervical cancer. In this study the correlation between size of lymph nodes and control doses of RT was analyzed. Between January 2002 and December 2007, 245 patients with squamous cell carcinoma of the cervix treated with a combination of external beam irradiation with or without boost irradiation and high-dose rate brachytherapy were investigated. Size of lymph node was measured by computed tomography before RT and just after 50 Gy RT. Of the 245 patients, 78 had PLN metastases, and a total of 129 had enlarged PLNs diagnosed as metastases; 22 patients had PLN failure. The PLN control rate at 5 years was 79.5% for positive cases and 95.8% for negative cases. In cases with positive PLNs, 12 of 129 nodes (9.3%) developed recurrences. There was significant correlation between PLN control rate and size of PLN after 50 Gy (<10 mm: 96.7%, ≥ 10 mm: 75.7 % (P<0.001)). In addition, the recurrence in these poor-response nodes was significantly correlated with dose of RT. Nine of 16 nodes receiving ≤ 58 Gy had recurrence, but none of 21 nodes receiving > 58 Gy had recurrence (P = 0.0003). These results suggested that the response of lymph nodes after RT was a more significant predictive factor for recurrence than size of lymph node before RT, and poor-response lymph nodes might require boost irradiation at a total dose of > 58 Gy.
PMCID: PMC3885130  PMID: 23912599
uterine cervical cancer; radiation therapy; boost irradiation; lymph node metastasis; pelvic lymph node
17.  Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds 
PLoS ONE  2013;8(6):e66220.
Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.
PMCID: PMC3670892  PMID: 23755301
18.  Genetic differences between Asian and Caucasian chronic lymphocytic leukemia 
International Journal of Oncology  2013;43(2):561-565.
Chronic lymphocytic leukemia (CLL) is a common hematological malignancy in Western countries. However, this disease is very rare in Asian countries. It is not clear whether the mechanisms of development of CLL in Caucasians and Asians are the same. We compared genetic abnormalities in Asian and Caucasian CLL using 250k GeneChip arrays. Both Asian and Caucasian CLL had four common genetic abnormalities: deletion of 13q14.3, trisomy 12, abnormalities of ATM (11q) and abnormalities of 17p. Interestingly, trisomy 12 and deletion of 13q14.3 were mutually exclusive in both groups. We also found that deletions of miR 34b/34c (11q), caspase 1/4/5 (11q), Rb1 (13q) and DLC1 (8p) are common in both ethnic groups. Asian CLL more frequently had gain of 3q and 18q. These suggest that classic genomic changes in the Asian and Caucasina CLL are same. Further, we found amplification of IRF4 and deletion of the SP140/SP100 genes; these genes have been reported as CLL-associated genes by previous genome-wide-association study. We have found classic genomic abnormalities in Asian CLL as well as novel genomic alteration in CLL.
PMCID: PMC3775563  PMID: 23708256
SNP-chip; common genomic changes; caspase; IRF4
19.  Intracellular mechanisms of aminoglycoside-induced cytotoxicity 
Since introduction into clinical practice over 60 years ago, aminoglycoside antibiotics remain important drugs in the treatment of bacterial infections, cystic fibrosis and tuberculosis. However, the ototoxic and nephrotoxic properties of these drugs are still a major clinical problem. Recent advances in molecular biology and biochemistry have begun to uncover the intracellular actions of aminoglycosides that lead to cytotoxicity. In this review, we discuss intracellular binding targets of aminoglycosides, highlighting specific aminoglycoside-binding proteins (HSP73, calreticulin and CLIMP-63) and their potential for triggering caspases and Bcl-2 signalling cascades that are involved in aminoglycoside-induced cytotoxicity. We also discuss potential strategies to reduce aminoglycoside cytotoxicity, which are necessary for greater bactericidal efficacy during aminoglycoside pharmacotherapy.
PMCID: PMC3662252  PMID: 21799993
20.  Cross-sectional study of correlation between mandibular incisor crowding and third molars in young Brazilians 
Objectives: The aim of this study was to evaluate transversally the clinical correlation between lower incisor crowding and mandible third molar. Study Design: Three hundred healthy volunteers (134 male and 166 female), aged 20.4 (±2.4) years-old were submitted to a complete clinical examination and filled up a questionnaire about gender, age, total teeth number and presence or absence of superior and inferior third molar. After a recent panoramic radiography were evaluated. The multiple logistic regression showed that none of the studied factors influenced the mandibular incisor crowding. Results: The proportion of both molars present or both absent was higher than the other conditions (Chi-square, p<.0001). The multiple logistic regression showed that any of the studied factors, influenced (p>.05) the mandibular incisor crowding. Despite the statistical significance, wear orthodontics appliances showed a little correlation (odds ratios < 1.0) in the mandibular incisor crowding. Conclusion: Presence of maxillary and/or mandibular third molars has no relation with the lower incisor crowding.
Key words:Malocclusion, third molars, lower incisor crowding, mandible.
PMCID: PMC3668880  PMID: 23385508
21.  Patterns of radiotherapy practice for biliary tract cancer in Japan: results of the Japanese radiation oncology study group (JROSG) survey 
The patterns of radiotherapy (RT) practice for biliary tract cancer (BTC) in Japan are not clearly established.
A questionnaire-based national survey of RT used for BTC treatment between 2000 and 2011 was conducted by the Japanese Radiation Oncology Study Group. Detailed information was collected for 555 patients from 31 radiation oncology institutions.
The median age of the patients was 69 years old (range, 33–90) and 81% had a good performance status (0–1). Regarding RT treatment, 78% of the patients were treated with external beam RT (EBRT) alone, 17% received intraluminal brachytherapy, and 5% were treated with intraoperative RT. There was no significant difference in the choice of treatment modality among the BTC subsites. Many patients with EBRT were treated with a total dose of 50 or 50.4 Gy (~40%) and only 13% received a total dose ≥60 Gy, even though most institutions (90%) were using CT-based treatment planning. The treatment field consisted of the primary tumor (bed) only in 75% of the patients. Chemotherapy was used for 260 patients (47%) and was most often administered during RT (64%, 167/260), followed by after RT (63%, 163/260). Gemcitabine was the most frequently used drug for chemotherapy.
This study established the general patterns of RT practice for BTC in Japan. Further surveys and comparisons with results from other countries are needed for development and optimization of RT for patients with BTC in Japan.
PMCID: PMC3622593  PMID: 23547715
Biliary tract cancer; Radiotherapy; Chemotherapy; Adjuvant; Palliative
22.  Clarifying the links between social support and health: Culture, stress, and neuroticism matter 
Journal of health psychology  2012;18(2):226-235.
Although it is commonly assumed that social support positively predicts health, the empirical evidence has been inconsistent. We argue that three moderating factors must be considered: (1) support-approving norms (cultural context); (2) support-requiring situations (stressful events); and (3) support-accepting personal style (low neuroticism). Our large-scale cross-cultural survey of Japanese and US adults found significant associations between perceived support and health. The association was more strongly evident among Japanese (from a support-approving cultural context) who reported high life stress (in a support-requiring situation). Moreover, the link between support and health was especially pronounced if these Japanese were low in neuroticism.
PMCID: PMC3556221  PMID: 22419414
culture; neuroticism; social support; stress
23.  Radiotherapy with fraction size of 2.25 Gy in T1-2 laryngeal and hypopharyngeal cancer 
Journal of Radiation Research  2013;54(4):684-689.
This study was carried out to evaluate the influence of fraction size 2.25 Gy on local control of T1 and T2 laryngeal and hypopharyngeal cancers. Between August 2002 and December 2010, 80 patients with T1 and T2 laryngeal or hypopharyngeal cancers were treated with definitive radiotherapy with a fraction size of 2.25 Gy. Primary sites were the larynx in 69 and the hypopharynx in 11. Fifty-three patients were T1 and 27 were T2. All patients' pathology was squamous cell carcinoma except one carcinosarcoma. Radiotherapy was delivered 5 days/week with a 4-MV photon beam up to a total dose of 63.0 Gy. Median treatment time was 41 days. Statistical analysis of survival was calculated using the Kaplan–Meier method. No acute toxicity greater than grade 2 (CTCAE ver. 3.0.) including mucositis and dermatitis was observed. All but one patient had a complete response. The partial response patient received salvage surgery. The median follow-up period was 47 months (ranging from 4 to 108 months). No late toxicity greater than 1 was observed. Nine patients developed recurrence, seven local and two neck lymph nodes. Three patients died, one from laryngeal cancer and two from intercurrent diseases. The 5-year local control rates (LCRs) in the entire group, larynx T1, larynx T2 and hypopharynx T1 were 85.8%, 97.6%, 70.1% and 85.7%, respectively. The LCRs of T1 improved compared with our historical control, but not those of T2. The 2.25-Gy fraction size is safe and may have the potential to achieve good LCR in T1 lesions.
PMCID: PMC3709663  PMID: 23297315
hypofractionated radiotherapy; laryngeal cancer; hypopharyngeal cancer
24.  Metabolome Analysis of Erythrocytes from Patients with Chronic Hepatitis C Reveals the Etiology of Ribavirin-Induced Hemolysis 
Ribavirin is one of the major agents used in combination therapy with interferon for chronic hepatitis C, but is often associated with hemolytic anemia as a serious adverse event. Employing metabolome analysis, we demonstrated that the concentrations of intermediate metabolites produced by glycolysis and the pentose phosphate cycle in patients' erythrocytes were significantly decreased after administration of ribavirin. Our findings suggest that hemolysis associated with ribavirin is triggered by an energy crisis and consequent oxidative stress, thus having implications for the prevention of such hemolysis.
PMCID: PMC3775117  PMID: 24046534
HCV; ribavirin; anemia; metabolome; glycolysis; pentose phosphate cycle.
25.  Identification of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs) as a Novel Target of Bisphenol A 
PLoS ONE  2012;7(12):e50481.
Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100–300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.
PMCID: PMC3515620  PMID: 23227178

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