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author:("matsuhashi, A")
1.  Critical Role of Exogenous Nitric Oxide in ROCK Activity in Vascular Smooth Muscle Cells 
PLoS ONE  2014;9(10):e109017.
Objective
Rho-associated kinase (ROCK) signaling pathway has been shown to mediate various cellular functions including cell proliferation, migration, adhesion, apoptosis, and contraction, all of which may be involved in pathogenesis of atherosclerosis. Endogenous nitric oxide (NO) is well known to have an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in vascular smooth muscle cells (VSMCs) in vitro and in vivo.
Methods
VSMCs migration was evaluated using a modified Boyden chamber assay. ROCK activities were measured by Western blot analysis in murine and human VSMCs and aorta of mice treated with or without angiotensin II (Ang II) and/or sodium nitroprusside (SNP), an NO donor.
Results
Co-treatment with SNP inhibited the Ang II-induced cell migration and increases in ROCK activity in murine and human VSMCs. Similarly, the increased ROCK activity 2 weeks after Ang II infusion in the mouse aorta was substantially inhibited by subcutaneous injection of SNP.
Conclusions
These findings suggest that administration of exogenous NO can inhibit ROCK activity in VSMCs in vitro and in vivo.
doi:10.1371/journal.pone.0109017
PMCID: PMC4184841  PMID: 25280018
2.  Effect of bevacizumab combined with boron neutron capture therapy on local tumor response and lung metastasis 
The aim of the present study was to evaluate the effect of bevacizumab on local tumor response and lung metastatic potential during boron neutron capture therapy (BNCT) and in particular, the response of intratumor quiescent (Q) cells. B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously administered bromodeoxyuridine (BrdU) to label all proliferating (P) tumor cells. The tumors were irradiated with thermal neutron beams following the administration of a 10B-carrier [L-para-boronophenylalanine-10B (BPA) or sodium mercaptoundecahydrododecaborate-10B (BSH)], with or without the administration of bevacizumab. This was further combined with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH, 40°C for 60 min). Immediately following the irradiation, cells from certain tumors were isolated and incubated with a cytokinesis blocker. The responses of the Q cells and the total (P+Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumor-bearing mice, 17 days following irradiation, lung metastases were enumerated. Three days following bevacizumab administration, the sensitivity of the total tumor cell population following BPA-BNCT had increased more than that following BSH-BNCT. The combination with MTH, but not with nicotinamide, further enhanced total tumor cell population sensitivity. Regardless of the presence of a 10B-carrier, MTH enhanced the sensitivity of the Q cell population. Regardless of irradiation, the administration of bevacizumab, as well as nicotinamide treatment, demonstrated certain potential in reducing the number of lung metastases especially in BPA-BNCT compared with BSH-BNCT. Thus, the current study revealed that BNCT combined with bevacizumab has the potential to sensitize total tumor cells and cause a reduction in the number of lung metastases to a similar level as nicotinamide.
doi:10.3892/etm.2014.1704
PMCID: PMC4061189  PMID: 24944637
bevacizumab; boron neutron capture therapy; 10B-carrier; quiescent cell; nicotinamide; mild temperature hyperthermia
3.  The dependency of compound biological effectiveness factors on the type and the concentration of administered neutron capture agents in boron neutron capture therapy 
SpringerPlus  2014;3:128.
Purpose
To examine the effect of the type and the concentration of neutron capture agents on the values of compound biological effectiveness (CBE) in boron neutron capture therapy.
Methods and materials
After the subcutaneous administration of a 10B-carrier, boronophenylalanine-10B (BPA) or sodium mercaptododecaborate-10B (BSH), at 3 separate concentrations, the 10B concentrations in tumors were measured by γ-ray spectrometry. SCC VII tumor-bearing C3H/He mice received 5-bromo-2′-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells, then treated with BPA or BSH. Immediately after reactor neutron beam irradiation, during which intratumor 10B concentrations were kept at levels similar to each other, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of BrdU-unlabeled quiescent (Q) and total (= P + Q) tumor cells were assessed based on the frequencies of micronucleation using immunofluorescence staining for BrdU.
Results
The CBE values were higher in Q cells and in the use of BPA than total cells and BSH, respectively. In addition, the higher the administered concentrations were, the smaller the CBE values became, with a clearer tendency in the use of BPA than BSH. The values for neutron capture agents that deliver into solid tumors more dependently on uptake capacity of tumor cells became more changeable.
Conclusion
Tumor characteristics, such as micro-environmental heterogeneity, stochastic genetic or epigenetic changes, or hierarchical organization of tumor cells, are thought to partially influence on the value of CBE, meaning that the CBE value itself may be one of the indices showing the degree of tumor heterogeneity.
doi:10.1186/2193-1801-3-128
PMCID: PMC4320213
Boron neutron capture therapy; Neutron capture agent; Relative biological effectiveness; Compound biological effectiveness; Tumor heterogeneity; Quiescent cell
4.  Radiosensitivity of pimonidazole-unlabelled intratumour quiescent cell population to γ-rays, accelerated carbon ion beams and boron neutron capture reaction 
The British Journal of Radiology  2013;86(1021):20120302.
Objective
To detect the radiosensitivity of intratumour quiescent (Q) cells unlabelled with pimonidazole to accelerated carbon ion beams and the boron neutron capture reaction (BNCR).
Methods
EL4 tumour-bearing C57BL/J mice received 5-bromo-29-deoxyuridine (BrdU) continuously to label all intratumour proliferating (P) cells. After the administration of pimonidazole, tumours were irradiated with c-rays, accelerated carbon ion beams or reactor neutron beams with the prior administration of a 10B-carrier. Responses of intratumour Q and total (P+Q) cell populations were assessed based on frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of pimonidazole-unlabelled tumour cells was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole.
Results
Following c-ray irradiation, the pimonidazole-unlabelled tumour cell fraction showed significantly enhanced radiosensitivity compared with the whole tumour cell fraction, more remarkably in the Q than total cell populations. However, a significantly greater decrease in radiosensitivity in the pimonidazole-unlabelled cell fraction, evaluated using a delayed assay or a decrease in radiation dose rate, was more clearly observed among the Q than total cells. These changes in radiosensitivity were suppressed following carbon ion beam and neutron beam-only irradiaton. In the BNCR, the use of a 10B-carrier, especially L-para-boronophenylalanine-10B, enhanced the sensitivity of the pimonidazole-unlabelled cells more clearly in the Q than total cells.
Conclusion
The radiosensitivity of the pimonidazole-unlabelled cell fraction depends on the quality of radiation delivered and characteristics of the 10B-carrier used in the BNCR.
Advances in knowledge
The pimonidazole-unlabelled subfraction of Q tumour cells may be a critical target in tumour control.
doi:10.1259/bjr.20120302
PMCID: PMC3615400  PMID: 23255546
5.  Rho-Associated Kinase (ROCK) Activity, Endothelial Function and Cardiovascular Risk Factors 
Arteriosclerosis, thrombosis, and vascular biology  2011;31(10):10.1161/ATVBAHA.111.227892.
Objective
Cardiovascular diseases are associated with chronic activation of Rho-associated kinases (ROCKs) and endothelial dysfunction. Both increased ROCK activity and endothelial dysfunction are thought to be closely associated with conventional cardiovascular risk factors. The purpose of this study was to determine the relationship between ROCK activity, endothelial function and cardiovascular risk factors.
Methods and Results
We evaluated ROCK activity in peripheral leukocytes by Western blot analysis and flow-mediated vasodilation by ultrasonography in 242 men who had no cardiovascular or cerebrovascular diseases (mean age, 40±10 yr; range, 20 to 73 yr). ROCK activity was defined as the ratio of phospho myosin-binding subunit on myosin light chain phosphatase to total myosin-binding subunit. Univariate regression analysis revealed that leukocyte ROCK activity significantly correlated with body mass index (r=0.29, P=0.003), systolic blood pressure (r=0.25, P=0.01), low-density lipoprotein cholesterol level (r=0.21, P=0.04), and Framingham risk factor score, a cumulative cardiovascular risk index for heart attack, (r=0.31, P<0.001) and that flow-mediated vasodilation significantly correlated with age (r=−0.23, P=0.02), body mass index (r=0.19, P=0.05), systolic blood pressure (r=−0.22, P=0.03), total cholesterol level (r=−0.21, P=0.04), low-density lipoprotein cholesterol level (r=−0.22, P=0.04), glucose level (r=−0.20, P=0.04), and Framingham risk factor score (r=−0.37, P<0.001). There was a significant correlation between leukocyte ROCK activity and flow-mediated vasodilation (r=−0.41, P<0.001). Multivariate analysis revealed that flow-mediated vasodilation was an independent predictor of leukocyte ROCK activity.
Conclusions
These findings suggest cumulative cardiovascular risk may enhance ROCK activity and endothelial dysfunction, leading to progression of cardiovascular diseases and outcomes.
doi:10.1161/ATVBAHA.111.227892
PMCID: PMC3852695  PMID: 21737782
Rho-associated kinases; endothelial function; Framingham risk factor score
6.  Hyperuricemia is independently associated with endothelial dysfunction in postmenopausal women but not in premenopausal women 
BMJ Open  2013;3(11):e003659.
Objectives
The purpose of this study was to determine the relationships between uric acid, endothelial function and cardiovascular risk factors and to investigate whether menopausal status was associated with the relationship between uric acid and endothelial function in women.
Design
Cross-sectional study.
Setting
3 general hospitals in Japan.
Participants
749 Japanese women aged 30–74 years recruited from people who underwent health-screening examinations with agreement for measurement of vascular function.
Measures
We measured serum concentrations of uric acid and flow-mediated vasodilation (FMD). Percentage of FMD (peak diameter−baseline diameter/baseline diameter) was used for analysis. Endothelial dysfunction was defined as FMD ≤4.90%, division point for the lowest tertile and the middle tertile of FMD. Menopause women were defined as participants without menstruation for over 1 year or participants with a history of hysterectomy or bilateral oophorectomy.
Results
Of the 749 participants, 368 (49.1%) were premenopausal women and 381 (50.9%) were postmenopausal women. Age, body mass index, systolic blood pressure, total cholesterol, triglycerides, glucose, estimated glomerular filtration rate and Framingham risk score were significantly correlated with serum uric acid level. FMD showed a gradual decrease in accordance with the serum uric acid level in the entire study population (<4 mg/dL, 6.85±3.65%; 4 to <5 mg/dL, 6.79±3.60%; 5 to <6 mg/dL, 6.24±3.58%; ≥6 mg/dL, 5.27±3.18%; p=0.01). Multivariate analysis revealed that uric acid was a significantly independent risk factor for endothelial dysfunction in postmenopausal women (OR 1.23, 95% CI 1.01 to 1.50), but not in premenopausal women.
Conclusions
These findings suggest that uric acid can be used as a risk marker of endothelial dysfunction in a female population, and particularly as an independent risk factor in postmenopausal women but not in premenopausal women.
Registration number of the study
UMIN000003409.
doi:10.1136/bmjopen-2013-003659
PMCID: PMC3822312  PMID: 24213096
Vascular Medicine; Pathology
7.  Measurement of Rho-Associated Kinase (ROCK) Activity in Humans: Validity of Leukocyte p-MBS/t-MBS in Comparison with Vascular Response to Fasudil 
Atherosclerosis  2010;214(1):117-121.
Background
Rho-associated kinases (ROCKs) have been shown to be involved in the pathogenesis of atherosclerosis. It is clinically important to estimate the degree of ROCK activity in humans. The purpose of this study was to confirm the validity of a leukocyte ROCK parameter as an index of ROCK activity in comparison with vascular response to a ROCK inhibitor.
Methods and Results
We evaluated the ratio of phospho myosin-binding subunit (p-MBS) on myosin light-chain phosphatase to total MBS in peripheral leukocytes by Western blot analysis and forearm blood flow (FBF) response to the ROCK inhibitor fasudil using strain-gauge plethysmography in 36 healthy subjects and 39 patients with cardiovascular diseases. Fasudil (3, 10, 30 μg/min) was infused intra-arterially for 5 minutes at each dose. Leukocyte p-MBS/total-MBS was higher in cardiovascular diseases than in healthy subjects (0.97±0.37 vs. 0.51±0.14; P=0.002). Fasudil increased FBF from 4.9±1.2 to 14.5±5.7 mL/min/100 mL tissue (P<0.0001) in patients with cardiovascular diseases, while fasudil did not alter FBF in healthy subjects. There was a significant relationship between leukocyte p-MBS/total-MBS and maximal FBF response to fasudil in all subjects (r=0.72, P<0.0001). There was also a significant correlation between p-MBS/total-MBS and maximal FBF response to fasudil in patients with cardiovascular diseases (r=0.59, P<0.0001). In healthy subjects, there was no significant correlation between the two parameters.
Conclusions
These findings suggest that assessment of leukocyte ROCK activity is minimally invasive and does not require pharmacologic intervention using ROCK inhibitors. Leukocyte p-MBS/total-MBS may be useful for evaluating ROCK activity in a clinical setting.
doi:10.1016/j.atherosclerosis.2010.10.005
PMCID: PMC3807089  PMID: 21035804
8.  Calcium Channel Blocker and Rho-associated Kinase Activity in Patients with Hypertension 
Journal of hypertension  2011;29(2):373-379.
Background
Rho-associated kinases (ROCKs) play an important role in Ca2+ sensitization and vascular resistance. Activation of ROCKs is associated with hypertension. The purpose of this study was to evaluate the effect of the calcium channel blocker amlodipine on ROCKs activity in patients with hypertension.
Methods
We evaluated ROCK activity in peripheral leukocytes by Western blot analysis in 651 patients with hypertension treated with antihypertensive agents, 28 untreated hypertensive patients and 28 healthy subjects, and the effects of treatment with amlodipine or losartan for 12 weeks on ROCK activity in 28 untreated hypertensive patients who were randomly divided into an amlodipine group (n=14) and a losartan group (n=14). ROCK activity was defined as the ratio of phospho myosin-binding subunit (MBS) on myosin light-chain phosphatase to total-MBS.
Results
Leukocyte ROCK activity was greater in untreated patients with essential hypertension than in the healthy subjects (0.84±0.24 vs. 0.61±0.18, P=0.03). In 651 patients with hypertension treated with antihypertensive agents, ROCK activity was significantly lower in the calcium channel blocker-treated group than in the groups treated with renin-angiotensin system inhibitors, diuretics, and β blockers (0.70±0.24 vs. 0.85±0.29, 0.83±0.24, and 0.86±0.31, P<0.05, respectively). ROCK activity after 4 weeks and 12 weeks of treatment was significantly decreased in the amlodipine group (0 weeks: 0.85±0.25, 4 weeks: 0.66±0.16, 12 weeks: 0.64±0.15, P<0.05, respectively) but not in the losartan group, while the antihypertensive effects were similar in the two groups.
Conclusion
These findings suggest that calcium channel blocker amlodipine inhibits ROCK activity in patients with hypertension.
doi:10.1097/HJH.0b013e328340902d
PMCID: PMC3807090  PMID: 21063203
9.  Development of a simple and rapid method of precisely identifying the position of 10B atoms in tissue: an improvement in standard alpha autoradiography 
Journal of Radiation Research  2013;55(2):373-380.
Boron neutron capture therapy (BNCT) can be utilized to selectively kill cancer cells using a boron compound that accumulates only in cancer cells and not in normal cells. Tumor-bearing animals treated by BNCT are routinely used to evaluate long-term antitumor effects of new boron compounds. Alpha-autoradiography is one of the methods employed in the evaluation of antitumor effects. However, a standard alpha-autoradiography cannot detect the microdistribution of 10B because of the difficulty associated with the superposition of a tissue sample image and etched pits on a track detector with the etching process. In order to observe the microdistribution of 10B, some special methods of alpha-autoradiography have been developed that make use of a special track detector, or the atomic force microscope combined with X-ray and UV light irradiation. In contrast, we propose, herein, a simple and rapid method of precisely identifying the position of 10B using the imaging process and the shape of etched pits, such as their circularity, without the need to use special track detectors or a microscope. A brief description of this method and its verification test are presented in this article. We have established a method of detecting the microdistribution of 10B with submicron deviation between the position of etched pits and the position of reaction in a tissue sample, for a given circularity of etched pits.
doi:10.1093/jrr/rrt110
PMCID: PMC3951073  PMID: 24142968
boron neutron capture therapy; boron compound; alpha-autoradiography; microdistribution
10.  Boron neutron capture therapy outcomes for advanced or recurrent head and neck cancer 
Journal of Radiation Research  2013;55(1):146-153.
We retrospectively review outcomes of applying boron neutron capture therapy (BNCT) to unresectable advanced or recurrent head and neck cancers. Patients who were treated with BNCT for either local recurrent or newly diagnosed unresectable head or neck cancers between December 2001 and September 2007 were included. Clinicopathological characteristics and clinical outcomes were retrieved from hospital records. Either a combination of borocaptate sodium and boronophenylalanine (BPA) or BPA alone were used as boron compounds. In all the treatment cases, the dose constraint was set to deliver a dose <10–12 Gy-eq to the skin or oral mucosa. There was a patient cohort of 62, with a median follow-up of 18.7 months (range, 0.7–40.8). A total of 87 BNCT procedures were performed. The overall response rate was 58% within 6 months after BNCT. The median survival time was 10.1 months from the time of BNCT. The 1- and 2-year overall survival (OS) rates were 43.1% and 24.2%, respectively. The major acute Grade 3 or 4 toxicities were hyperamylasemia (38.6%), fatigue (6.5%), mucositis/stomatitis (9.7%) and pain (9.7%), all of which were manageable. Three patients died of treatment-related toxicity. Three patients experienced carotid artery hemorrhage, two of whom had coexistent infection of the carotid artery. This study confirmed the feasibility of our dose-estimation method and that controlled trials are warranted.
doi:10.1093/jrr/rrt098
PMCID: PMC3885131  PMID: 23955053
boron neutron capture therapy; head and neck tumors
11.  Autologous Bone-Marrow Mesenchymal Stem Cell Implantation and Endothelial Function in a Rabbit Ischemic Limb Model 
PLoS ONE  2013;8(7):e67739.
Background
The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model.
Methods
We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 106 MSCs were implanted into each ischemic limb.
Results
Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of NG-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar.
Conclusions
These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.
doi:10.1371/journal.pone.0067739
PMCID: PMC3701528  PMID: 23861797
12.  Effects of employing a 10B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy 
The British Journal of Radiology  2012;85(1011):249-258.
Objectives
To evaluate the effects of employing a 10B-carrier and manipulating intratumour hypoxia on local tumour response and lung metastatic potential in boron neutron capture therapy (BNCT) by measuring the response of intratumour quiescent (Q) cells.
Methods
B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumours received reactor thermal neutron beam irradiation following the administration of a 10B-carrier [L-para-boronophenylalanine-10B (BPA) or sodium mercaptoundecahydrododecaborate-10B (BSH)] in combination with an acute hypoxia-releasing agent (nicotinamide) or mild temperature hyperthermia (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P+Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.
Results
BPA-BNCT increased the sensitivity of the total tumour cell population more than BSH-BNCT. However, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. With or without a 10B–carrier, MTH enhanced the sensitivity of the Q cell population. Without irradiation, nicotinamide treatment decreased the number of lung metastases. With irradiation, BPA-BNCT, especially in combination with nicotinamide treatment, showed the potential to reduce the number of metastases more than BSH-BNCT.
Conclusion
BSH-BNCT in combination with MTH improves local tumour control, while BPA-BNCT in combination with nicotinamide may reduce the number of lung metastases.
doi:10.1259/bjr/20974899
PMCID: PMC3473983  PMID: 22391496
13.  Reducing intratumour acute hypoxia through bevacizumab treatment, referring to the response of quiescent tumour cells and metastatic potential 
The British Journal of Radiology  2011;84(1008):1131-1138.
Objectives
The aim was to evaluate the influence of bevacizumab on intratumour oxygenation status and lung metastasis following radiotherapy, with specific reference to the response of quiescent (Q) cell populations within irradiated tumours.
Methods
B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation following treatment with the acute hypoxia-releasing agent nicotinamide or local mild temperature hyperthermia (MTH) with or without the administration of bevacizumab under aerobic conditions or totally hypoxic conditions, achieved by clamping the proximal end of the tumours. Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In the other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation.
Results
3 days after bevacizumab administration, acute hypoxia-rich total cell population in the tumour showed a remarkably enhanced radiosensitivity to γ-rays, and the hypoxic fraction (HF) was reduced, even after MTH treatment. However, the hypoxic fraction was not reduced after nicotinamide treatment. With or without γ-ray irradiation, bevacizumab administration showed some potential to reduce the number of lung metastases as well as nicotinamide treatment.
Conclusion
Bevacizumab has the potential to reduce perfusion-limited acute hypoxia and some potential to cause a decrease in the number of lung metastases as well as nicotinamide.
doi:10.1259/bjr/38457938
PMCID: PMC3473837  PMID: 21586505
14.  Wortmannin efficiently suppresses the recovery from radiation-induced damage in pimonidazole-unlabeled quiescent tumor cell population 
Journal of Radiation Research  2012;54(2):221-229.
Labeling of proliferating (P) cells in mice bearing EL4 tumors was achieved by continuous administration of 5-bromo-2′-deoxyuridine (BrdU). Tumors were irradiated with γ-rays at 1 h after pimonidazole administration followed by caffeine or wortmannin treatment. Twenty-four hours later, assessment of the responses of quiescent (Q) and total (= P + Q) cell populations were based on the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of the pimonidazole-unlabeled tumor cell fractions was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole. The pimonidazole-unlabeled cell fraction showed significantly enhanced radio-sensitivity compared with the whole cell fraction more remarkably in Q cells than total cells. However, a significantly greater decrease in radio-sensitivity in the pimonidazole-unlabeled than the whole cell fraction, evaluated using an assay performed 24 hours after irradiation, was more clearly observed in Q cells than total cells. In both the pimonidazole-unlabeled and the whole cell fractions, wortmannin efficiently suppressed the reduction in sensitivity due to delayed assay. Wortmannin combined with γ-ray irradiation is useful for suppressing the recovery from radiation-induced damage especially in the pimonidazole-unlabeled cell fraction within the total and Q tumor cell populations.
doi:10.1093/jrr/rrs094
PMCID: PMC3589932  PMID: 23097299
Quiescent cell; recovery from radiation-induced damage; pimonidazole; wortmannin; caffeine
15.  Significance of manipulating tumour hypoxia and radiation dose rate in terms of local tumour response and lung metastatic potential, referring to the response of quiescent cell populations 
The British Journal of Radiology  2010;83(993):776-784.
The purpose of this study was to evaluate the influence of manipulating intratumour oxygenation status and radiation dose rate on local tumour response and lung metastases following radiotherapy, referring to the response of quiescent cell populations within irradiated tumours. B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating (P) cells. They received γ-ray irradiation at high dose rate (HDR) or reduced dose rate (RDR) following treatment with the acute hypoxia-releasing agent nicotinamide or local hyperthermia at mild temperatures (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the quiescent (Q) and total (proliferating + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Following HDR irradiation, nicotinamide and MTH enhanced the sensitivity of the total and Q-cell populations, respectively. The decrease in sensitivity at RDR irradiation compared with HDR irradiation was slightly inhibited by MTH, especially in Q cells. Without γ-ray irradiation, nicotinamide treatment tended to reduce the number of lung metastases. With γ-rays, in combination with nicotinamide or MTH, especially the former, HDR irradiation decreased the number of metastases more remarkably than RDR irradiation. Manipulating both tumour hypoxia and irradiation dose rate have the potential to influence lung metastasis. The combination with the acute hypoxia-releasing agent nicotinamide may be more promising in HDR than RDR irradiation in terms of reducing the number of lung metastases.
doi:10.1259/bjr/57015642
PMCID: PMC3473411  PMID: 20739345
16.  Torsade de pointes associated with bradycardia and takotsubo cardiomyopathy 
Two cases of torsade de pointes associated with bradycardia and takotsubo cardiomyopathy are reported. In both cases, atrioventricular block preceded the occurrence of takotsubo cardiomyopathy. Bradycardia-induced QT interval prolongation seemed to be amplified by the occurrence of takotsubo cardiomyopathy, resulting in torsade de pointes. Temporary ventricular pacing at a high rate decreased the QT interval and prevented the recurrence of torsade de pointes. Because atrioventricular block recurred or persisted even after the resolution of takotsubo cardiomyopathy, the patients received permanent pacemakers.
PMCID: PMC2644362  PMID: 18685745
Atrioventricular block; Takotsubo cardiomyopathy; Torsade de pointes
17.  Japanese study to organize proper lifestyle modifications for metabolic syndrome (J-STOP-MetS): Design and method 
Prevalence of the metabolic syndrome is now a very serious health problem in Japan and a public preventive strategy is essential to reduce morbidity. A systematic interventional strategy for the metabolic syndrome remains to be established. In order to address this issue, a multi-center study; Japanese Study to Organize Proper lifestyle modification for the metabolic syndrome (J-STOP-MetS), has been established by nine preventive medical centers among Rosai hospital groups. This study comprises a cross-sectional study (J-STOP-MetS 1) and a prospective randomized control study (J-STOP-MetS 2). J-STOP-MetS 1 examines the causes of the metabolic syndrome by means of a questionnaire in a large cohort of patients with the metabolic syndrome and control subjects matched for age and sex. J-STOP-MetS 2 examines the hypothesis that guidance on lifestyle modifications will help at risk patients to reduce abdominal fat and cardiovascular risk factors. The metabolic syndrome patients are randomly assigned either to a single visit to a guidance group or multiple visits every two months. The individualized guidance is provided by the coordination of physician, trained nurse, dietician and exercise trainer. Several parameters are measured before and six months after the first guidance session, including, body weight, waist circumference, blood pressure, several blood markers and arterial stiffness. The J-STOP-MetS is the first large-scale clinical study of the metabolic syndrome in Japan and should provide important evidence for the practical management of the metabolic syndrome.
PMCID: PMC2496971  PMID: 18561516
metabolic syndrome; J-STOP-MetS; hypertension; diabetes; dyslipidemia
18.  Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs 
Nucleic Acids Research  2007;36(2):616-628.
The nuclear RNA export factor (NXF) family proteins have been implicated in various aspects of post-transcriptional gene expression. This study shows that mouse NXF7 exhibits heterologous localization, i.e. NXF7 associates with translating ribosomes, stress granules (SGs) and processing bodies (P-bodies), the latter two of which are believed to be cytoplasmic sites of storage, degradation and/or sorting of mRNAs. By yeast two-hybrid screening, a series of heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as possible binding partners for NXF7. Among them, hnRNP A3, which is believed to be involved in translational control and/or cytoplasmic localization of certain mRNAs, formed a stable complex with NXF7 in vitro. Although hnRNP A3 was not associated with translating ribosomes, it was co-localized with NXF7 in P-bodies. After exposing to oxidative stress, NXF7 trans-localized to SGs, whereas hnRNP A3 did not. In differentiated neuroblastoma Neuro2a cells, NXF7 was co-localized with hnRNP A3 in cell body and neurites. The amino terminal half of NXF7, which was required for stable complex formation with hnRNP A3, coincided with the region required for localization in both P-bodies and neuronal RNA granules. These findings suggest that NXF7 plays a role in sorting, transport and/or storage of mRNAs through interactions with hnRNP A3.
doi:10.1093/nar/gkm556
PMCID: PMC2241847  PMID: 18063567
19.  Relationship between flow-mediated vasodilation and cardiovascular risk factors in a large community-based study 
Heart  2013;99(24):1837-1842.
Objective
To determine the relationships between flow-mediated vasodilation (FMD) and cardiovascular risk factors, and to evaluate confounding factors for measurement of FMD in a large general population in Japan.
Methods
This was a cross-sectional study. A total of 5314 Japanese adults recruited from people who underwent health screening from 1 April 2010 to 31 August 2012 at 3 general hospitals in Japan. Patients’ risk factors (age, Body Mass Index, blood pressure, cholesterol parameters, glucose level and HbA1c level) and prevalence of cardiovascular disease (coronary heart disease and cerebrovascular disease) were investigated.
Results
Univariate regression analysis revealed that FMD correlated with age (r=−0.27, p<0.001), Body Mass Index (r=−0.14, p<0.001), systolic blood pressure (r=−0.18, p<0.001), diastolic blood pressure (r=−0.13, p<0.001), total cholesterol (r=−0.07, p<0.001), triglycerides (r=−0.10, p<0.001), high-density lipoprotein cholesterol (r=0.06, p<0.001), low-density lipoprotein cholesterol (r=−0.04, p=0.01), glucose level (r=−0.14, p<0.001), HbA1c (r=−0.14, p<0.001), and baseline brachial artery diameter (r=−0.43, p<0.001) as well as Framingham Risk score (r=−0.29, p<0.001). Multivariate analysis revealed that age (t value=−9.17, p<0.001), sex (t value=9.29, p<0.001), Body Mass Index (t value=4.27, p<0.001), systolic blood pressure (t value=−2.86, p=0.004), diabetes mellitus (t value=−4.19, p<0.001), smoking (t value=−2.56, p=0.01), and baseline brachial artery diameter (t value=−29.4, p<0.001) were independent predictors of FMD.
Conclusions
FMD may be a marker of the grade of atherosclerosis and may be used as a surrogate marker of cardiovascular outcomes. Age, sex, Body Mass Index, systolic blood pressure, diabetes mellitus, smoking and, particularly, baseline brachial artery diameter are potential confounding factors in the measurement of FMD.
doi:10.1136/heartjnl-2013-304739
PMCID: PMC3841746  PMID: 24153417
20.  The Ionic Permeability Changes during Acetylcholine-Induced Responses of Aplysia Ganglion Cells 
The Journal of General Physiology  1968;51(3):321-345.
ACh-induced depolarization (D response) in D cells markedly decreases as the external Na+ is reduced. However, when Na+ is completely replaced with Mg++, the D response remains unchanged. When Na+ is replaced with Tris(hydroxymethyl)aminomethane, the D response completely disappears, except for a slight decrease in membrane resistance. ACh-induced hyperpolarization (H response) in H cells is markedly depressed as the external Cl- is reduced. Frequently, the reversal of the H response; i.e., depolarization, is observed during perfusion with Cl--free media. In cells which show both D and H responses superimposed, it was possible to separate these responses from each other by perfusing the cells with either Na+-free or Cl--free Ringer's solution. High [K+]0 often caused a marked hyperpolarization in either D or H cells. This is due to the primary effect of high [K+]0 on the presynaptic inhibitory fibers. The removal of this inhibitory afferent interference by applying Nembutal readily disclosed the predicted K+ depolarization. In perfusates containing normal [Na+]0, the effects of Ca++ and Mg++ on the activities of postsynaptic membrane were minimal, supporting the current theory that the effects of these ions on the synaptic transmission are mainly presynaptic. The possible mechanism of the hyperpolarization produced by simultaneous perfusion with both high [K+]0 and ACh in certain H cells is explained quantitatively under the assumption that ACh induces exclusively an increase in Cl- permeability of the H membrane.
PMCID: PMC2201135  PMID: 5648831

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