Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Uniwersytet Jagiellonski - Wydawnictwo Uniwersytetu Jagiellonskiego  

DOI： 10.4467/16890027ap.20.001.12157

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The cochlear stria vascularis produces endolymph and thereby plays an active role in inner ear homeostasis. We recently reported that the H+/myo-inositol cotransporter (HMIT) gene is expressed in the stria vascularis. Here, we examined the protein localization of HMIT and Na+/myo-inositol cotransporter 1 (SMIT1) in the stria vascularis by immunohistochemistry. HMIT and SMIT1 were detected in the lateral wall of the cochlear duct. HMIT was widely detected throughout the stria vascularis, while SMIT1 was enriched in the strial basal cells. To examine the localization of HMIT in the stria vascularis in more detail, dissociated strial cells were immunostained, which resulted in the detection of HMIT immunoreactivity in marginal cells. These results indicate that HMIT is expressed in marginal cells and basal cells of the stria vascularis, while SMIT1 expression is enriched in basal cells. We speculate that HMIT and SMIT1 may play important roles in the homeostasis of cochlear fluids, for example by participating in pH regulation and osmoregulation.

DOI： 10.1016/j.neulet.2018.03.028

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Despite the importance of glucose as an energy source for marginal cells of the cochlear stria vascularis, no direct evidence of the glucose uptake into these cells has been demonstrated. In this study, we investigated the glucose pathway in strial marginal cells using a fluorescent tracer method combined with immunohistochemistry. For glucose imaging, a non-metabolizable fluorescent glucose analog, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG), was intravenously injected into mice. After 6-NBDG fluorescent signals in cochlear sections were observed, the same sections were processed for immunohistochemistry to identify marginal cells. This showed that 6-NBDG fluorescent signals had passed into the cytosol of marginal cells via Na⁺/K⁺-ATPase-positive basolateral membranes. These results provide direct physiological evidence of the glucose uptake into epithelial strial marginal cells. This combined method can help in examining the relationship between target molecules and functional proteins using the same tissue samples.

DOI： 10.11169/bioimages.23.1

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ACh Prevents Ischemic Loss of Gj and Arrhythmias
IntroductionAcetylcholine (ACh), a vagal efferent neurotransmitter, markedly improves survival in rats with myocardial ischemia (MI) by preventing ischemic loss of gap junction (Gj) and by inducing anti-apoptotic cascades. However, electrophysiological mechanisms of the antiarrhythmic effect of ACh after acute MI are still unclear.
MethodsAcute MI was induced by ligation of the left anterior descending (LAD) coronary artery in Langendorff-perfused rabbit hearts with (ACh(+):n = 11) or without (ACh(-):n = 12) 10 mol/L ACh delivered continuously starting at 5 minutes before LAD ligation. Action potentials on the left ventricular (LV) anterior surface (approximate to 2x2 cm) were recorded by optical mapping during pacing from the LV epicardium (BCL = 500 milliseconds). Conduction velocities (CVs) at 256 sites were calculated and the ventricular tachycardia/ventricular fibrillation (VT/VF) susceptibility was also assessed by programmed electrical stimulation before and 30 minutes after MI. The amount and distribution of Gj protein connexin-43 was analyzed by immunoblotting and immunohistochemistry.
ResultsAveraged CV in the ischemic border zone (IBZ) was significantly slower in ACh(-) than in ACh(+) (21 7 vs. 34 +/- 6 cm/s; P < 0.01). Short-coupled extra stimulus further decreased CV of IBZ in ACh(-) (13 +/- 4 cm/s) but did not change that in ACh(+) (34 +/- 5 cm/s), leading to a high incidence of conduction block in IBZ in ACh(-) but not in ACh(+) (83% vs. 0%). VT/VF after MI were induced in ACh(-) but suppressed in ACh(+) (10/12 vs. 3/11; P < 0.01). Connexin-43 in the LV anterior wall was significantly reduced after MI in ACh(-) but not in ACh(+).
ConclusionACh may suppress VT/VF by preventing loss of Gj and improving CV in IBZ during acute MI.

DOI： 10.1111/jce.12663

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Authorship：Corresponding author   Language：Japanese  

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DOI： 10.11169/bioimages.20.9

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

We have investigated the gene expression of the facilitated glucose transporter (GLUT), H(+)-coupled myoinositol cotransporter (HMIT), and Na(+) glucose cotransporter (SGLT) in the lateral wall of the cochlear duct by conventional RT-PCR and quantitative real-time PCR. The isoforms GLUT1, -3, -4, -5, -8, -10, -12 and HMIT were detected in both the stria vascularis and the spiral ligament, whereas no SGLT isoforms could be detected in these tissues. Quantitative real-time PCR analysis revealed significant differences in the gene expression of GLUT1, -4, -5, -10, and HMIT isoforms between the stria vascularis and the spiral ligament. This result reflects the tissue-dependent distributions of GLUT isoforms. These findings strongly suggest that a number of GLUT isoforms participate in glucose transport in the stria vascularis and the spiral ligament. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2010.12.029

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Vagal nerve stimulation (VS) has been reported to improve the survival after both acute and chronic myocardial infarction through the release of neurotransmitter ACh. However, the precise mechanism behind its beneficial effect is still unknown. In this study, we demonstrate the upregulation of tumor necrosis factor-alpha (TNF-alpha) and its cell survival TNF receptor-2 (TNFR2) as the mechanism behind VS induced myocardial protection. We investigated the effects of efferent VS on myocardial ischemic injury with in vivo and in vitro mouse models. In in vivo hearts VS significantly increased the expression of TNF-alpha both at the messenger and protein level after 3-hours of myocardial ischemia. In the in vitro studies ACh treatment before hypoxia, induced a significant upregulation of TNF-alpha compared to the untreated cardiomyocytes. Immunofluorescence analysis confirmed the synthesis of TNF-alpha by cardiomyocytes both in vivo and in vitro. VS also significantly reduced the myocardial infarct size (23.9 +/- 5.7% vs. 56 +/- 1.9%) and activated the cell survival Akt cascade system. Further, ACh upregulated the cell survival TNFR2 expression, while downregulating the cell destructive TNF receptor 1 (TNFR1) expression. These results were confirmed using the TNF receptors deficient mice, where the VS mediated protection was lost both in vivo and in vitro in TNFR2 (TNFR2(-/-)) and TNF receptors double knock out (TNFR1(-/-)2(-/-)) mice. VS and ACh protects the heart against acute ischemia or hypoxic injury by differentially regulating the TNF receptor subtypes. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.yjmcc.2010.03.007

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Background: Understanding the basic mechanisms and prevention of any disease pattern lies mainly on development of a successful experimental model. Recently, engineered heart tissue (EHT) has been demonstrated to be a useful tool in experimental transplantation. Here, we demonstrate a novel function for the spontaneously contracting EHT as an experimental model in studying the acute ischemia-induced changes in vitro.
Methodology/Principal Findings: EHT was constructed by mixing cardiomyocytes isolated from the neonatal rats and cultured in a ring-shaped scaffold for five days. This was followed by mechanical stretching of the EHT for another one week under incubation. Fully developed EHT was subjected to hypoxia with 1% O(2) for 6 hours after treating them with cell protective agents such as cyclosporine A (CsA) and acetylcholine (ACh). During culture, EHT started to show spontaneous contractions that became more synchronous following mechanical stretching. This was confirmed by the increased expression of gap junctional protein connexin 43 and improved action potential recordings using an optical mapping system after mechanical stretching. When subjected to hypoxia, EHT demonstrated conduction defects, dephosphorylation of connexin-43, and down-regulation of cell survival proteins identical to the adult heart. These effects were inhibited by treating the EHT with cell protective agents.
Conclusions/Significance: Under hypoxic conditions, the EHT responds similarly to the adult myocardium, thus making EHT a promising material for the study of cardiac functions in vitro.

DOI： 10.1371/journal.pone.0009275

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE INC MEDICAL PUBLISHERS  

Background: We previously reported that chronic vagal nerve Stimulation markedly improved long-term survival after chronic heart failure (CHF) in rats through cardioprotective effects of acetylcholine, independent of the heart rate-slowing mechanism. However, such ail approach is invasive and its safety is unknown in clinical settings. To develop an alternative therapy with a clinically available drug, we examined the chronic effect of oral donepezil, in acetylcholinesterase inhibitor against Alzheimer's disease, on cardiac remodeling and survival with a murine model of volume-overloaded CHF.
Methods and Results: Four weeks after surgery of aortocaval shunt, CHF mice were randomized into untreated and donepezil-treated groups. Donepezil was orally given at a dosage of 5 mg.kg(-1).day(-1). After 4 weeks of treatment, we evaluated in situ left ventricular (LV) pressure, ex vivo LV pressure-volume relationships, and LV expression of brain natriuretic peptides (BNP). We also observed Survival for 50 days. When compared with the untreated group, the donepezil-treated group had significantly low LV end-diastolic pressure, high LV contractility, and low LV expression of BNP. Donepezil significantly reduced the heart weight and markedly improved the Survival rate during the 50-day treatment period (54% versus 81%, P < .05).
Conclusions: Oral donepezil improves Survival of CHF mice through prevention of pumping failure and cardiac remodeling. (J Cardiac Fail 2009;15:805-811)

DOI： 10.1016/j.cardfail.2009.05.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOSBY-ELSEVIER  

Background: In spite of recent advances in coronary interventional therapy, reperfusion injury is still considered to be a major problem in patients undergoing surgical procedures, such as bypass grafting. Here we demonstrate a novel therapeutic strategy against ischemia-reperfusion injury: vagally mediated prevention of reperfusion-induced opening of mitochondrial permeability transition pore.
Methods: We investigated the effects of efferent vagal stimulation on myocardial reperfusion injury with ex vivo and in vitro rat models. In the ex vivo model the hearts were perfused with intact vagal innervation, which allowed us to study the effects of the vagal nerve on the heart without other systemic effects.
Results: Compared with sham stimulation, vagal stimulation exerted a marked anti-infarct effect irrespective of the heart rate (34% +/- 6% vs 85% +/- 9% at a heart rate of 300 beats/min, 37% +/- 4% vs 43% +/- 5% at a heart rate of 250 beats/min, and 39% +/- 4% vs 88% +/- 7% at a heart rate of 350 beats/min) after a 30-minute period of global ischemia, activated cell-survival Akt cascade, prevented downregulation of the antiapoptotic protein Bcl-2, and suppressed cytochrome-c release and caspase-3 activation. Furthermore, vagal stimulation-treated hearts exhibited a significant improvement in left ventricular developed pressure (78 +/- 5 vs 45 +/- 8 mm Hg) and a significant attenuation in an incremental change in left ventricular end-diastolic pressure during reperfusion. These beneficial effects of vagal stimulation were abolished by a permeability transition pore opener, atractyloside. In the in vitro study with primary-cultured cardiomyocytes, acetylcholine prevented a reoxygenation-induced collapse in mitochondrial transmembrane potential through inhibition of permeability transition pore opening.
Conclusion: Vagal stimulation would be a potential adjuvant therapy for the rescue of ischemic myocardium from reperfusion injury, and the protective effects are independent of its bradycardiac effects.

DOI： 10.1016/j.jtcvs.2008.08.020

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Language：Japanese   Publisher：Japan Heart Foundation  

DOI： 10.11281/shinzo.41.115

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Other Link： http://search.jamas.or.jp/link/ui/2009128118

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Immunoreactivity for the facilitated glucose transporter 1 (GLUT-1) has been found in the cochlear stria vascularis, but whether the strial marginal cells are immunopositive for GLUT-1 remains uncertain. To determine the cellular localization of GLUT-1 and to clarify the glucose pathway in the stria vascularis of rats and guinea pigs, immunohistochemistry was performed on sections, dissociated cells, and whole-tissue preparations. Immunoreactivity for GLUT-1 in sections was observed in the basal side of the strial tissue and in capillaries in both rats and guinea pigs. However, the distribution of the positive signals within the guinea pig strial tissue was more diffuse than that in rats. Immunostaining of dissociated guinea pig strial cells revealed GLUT-1 in the basal cells and capillary endothelial cells, but not in the marginal cells. These results indicated that GLUT-1 was not expressed in the marginal cells, and that another isoform of GLUT was probably expressed in these cells. Three-dimensional observation of whole-tissue preparations demonstrated that cytoplasmic prolongations from basal cells extended upward to the apical surface of the stria vascularis from rats and guinea pigs, and that the marginal cells were surrounded by these protrusions. We speculate that these upward extensions of basal cells have been interpreted as basal infoldings of marginal cells in previous reports from other groups. The three-dimensional relationship between marginal cells and basal cells might contribute to the transcellular glucose pathway from perilymph to intrastrial space.

DOI： 10.1007/s00441-007-0495-2

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Authorship：Lead author   Language：Japanese   Publisher：公益財団法人 日本心臓財団  

DOI： 10.11281/shinzo1969.40.Supplement2_34

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Other Link： http://search.jamas.or.jp/link/ui/2008283508

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Our previous study reveals that connexin (Cx) 43 is targeted by ACh to prevent lethal arrhythmia. Granulocyte colony-stimulating factor (G-CSF), used against ischemic heart failure, may be another candidate, however, with unknown mechanisms. Therefore, we investigated the cellular effects of G-CSF. G-CSF activated the Wnt and Jak2 signals in cardiomyocytes, and up-regulated Cx43 protein and phosphorylation levels. In addition, G-CSF enhanced the localization of Cx43, beta-catenin and cadherin on the plasma membrane. G-CSF inhibited the reduction of Cx43 by enhancing Cx43 anchoring and sustained the cell-cell communication during hypoxia. Consequently, GCSF suppressed ventricular arrhythmia induced by myocardial infarction. As a result, G-CSF could be used as a therapeutic tool for arrhythmia. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2007.09.007

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Authorship：Lead author   Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

In a recent study, we demonstrated that vagal stimulation increases the survival of rats with myocardial infarction by inhibiting lethal arrhythmia through regulation of connexin43 (Cx43). However, the precise mechanisms for this effect remain to be elucidated. To investigate these mechanisms and the signal transduction for gap junction regulation, we investigated the effect of acetylcholine (ACh), a parasympathetic nerve system neurotransmitter, on the gap junction component Cx43 using H9c2 cells. When cells were subjected to hypoxia, the total Cx43 protein level was decreased. In contrast, pretreatment with ACh inhibited this effect. To investigate the signal transduction, cells were pretreated with L-NAME, a nitric oxide synthase inhibitor, followed by ACh and hypoxia. L-NAME was found to suppress the ACh effect. However, a NO donor, SNAP, partially inhibited the hypoxia-induced reduction in Cx43. To delineate the mechanisms of the decrease in Cx43 under hypoxia, cells were pretreated with MG132, a proteasome inhibitor. Proteasome inhibition produced a striking recovery of the decrease in the total Cx43 protein level under hypoxia. However, cotreatment with MG132 and ACh did not produce any further increase in the total Cx43 protein level. Functional studies using ACh or okadaic acid, a phosphatase inhibitor, revealed that both reagents inhibited the decrease in the dye transfer induced by hypoxia. These results suggest that ACh is responsible for restoring the decrease in the Cx43 protein level, resulting in functional activation of gap junctions.

DOI： 10.1254/jphs.FP0051023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHYSIOLOGICAL SOCIETY JAPAN  

Background: Hypoxia-inducible factor (HIF)-1 alpha regulates the transcription of lines of genes, including vascular endothelial growth factor (VEGF), a major gene responsible for angiogenesis. Several recent studies have demonstrated that a nonhypoxic pathway via nitric oxide (NO) is involved in the activation of HIF-1 alpha. However, there is no direct evidence demonstrating the release of angiogenic factors by cardiomyocytes through the nonhypoxic induction pathway of HIF-1 alpha in the heart. Therefore we assessed the effects of an NO donor, S-Nitroso-N-acetylpenicillamine (SNAP) on the induction of VEGF via HIF-1 alpha under normoxia, using primary cultured rat cardiomyocytes (PRCMs). Methods and Results: PRCMs treated with acetylcholine (ACh) or SNAP exhibited a significant production of NO. SNAP activated the induction of HIF-1 alpha protein expression in PRCMs during normoxia. Phosphatidlylinositol 3-kinase (PI3K)-dependent Akt phosphorylation was induced by SNAP and was completely blocked by wortmannin, a PI3K inhibitor, and N-G-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor. The SNAP treatment also increased VEGF protein expression in PRCMs. Furthermore, conditioned medium derived from SNAP-treated cardiomyocytes phosphorylated the VEGF type-2 receptor (Flk-1) of human umbilical vein endothelial cells (a fourfold increase compared to the control group, p < 0.001, n = 5) and accelerated angiogenesis. Conclusion: Our results suggest that cardiomyocytes produce VEGF through a nonhypoxic HIF-1 alpha induction pathway activated by NO, resulting in angiogenesis.

DOI： 10.2170/physiolsci.RP002305

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Background - We proposed a novel therapeutic strategy against central baroreflex failure: implementation of an artificial baroreflex system to automatically regulate sympathetic vasomotor tone, ie, a bionic baroreflex system (BBS), and we tested its efficacy in a model of sudden hypotension during surgery.
Methods and Results - The BBS consisted of a computer-controlled negative-feedback circuit that sensed arterial pressure (AP) and automatically computed the frequency (STM) of a pulse train required to stimulate sympathetic nerves via an epidural catheter placed at the level of the lower thoracic spinal cord. An operation rule was subsequently designed for the BBS using a feedback correction with proportional and integral gain factors. The transfer function from STM to AP was identified by a white noise system identification method in 12 sevoflurane-anesthetized patients undergoing orthopedic surgery involving the cervical vertebrae, and the feedback correction factors were determined with a numerical simulation to enable the BBS to quickly and stably attenuate an external disturbance on AP. The performance of the designed BBS was then examined in a model of orthostatic hypotension during knee joint surgery (n = 21). Without the implementation of the BBS, a sudden deflation of a thigh tourniquet resulted in a 17 +/- 3 mm Hg decrease in AP within 10 seconds and a 25 +/- 2 mm Hg decrease in AP within 50 seconds. By contrast, during real-time execution of the BBS, the decrease in AP was 9 +/- 2 mm Hg at 10 seconds and 1 +/- 2 mm Hg at 50 seconds after the deflation.
Conclusions - These results suggest the feasibility of a BBS approach for central baroreflex failure.

DOI： 10.1161/CIRCULATIONAHA.105.587915

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Language：Japanese   Publisher：JAPANESE SOCIETY FOR ARTIFICIAL ORGANS  

DOI： 10.11392/jsao1972.34.2Supplement_s40

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Authorship：Lead author   Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Background - Myocardial ischemia (MI) leads to derangements in cellular electrical stability and the generation of lethal arrhythmias. Vagal nerve stimulation has been postulated to contribute to the antifibrillatory effect. Here, we suggest a novel mechanism for the antiarrhythmogenic properties of vagal stimulation during acute MI.
Methods and Results - Under anesthesia, Wistar rats underwent 30 minutes of left coronary artery (LCA) ligation with vagal stimulation (MI-VS group, n = 11) and with sham stimulation (MI-SS group, n = 12). Eight of the 12 rats in the MI-SS group had ventricular tachyarrhythmia (VT) during 30-minute LCA ligation; on the other hand, VT occurred in only 1 of the 11 rats in the MI-VS group (67% versus 9%, respectively). Atropine administration abolished the antiarrhythmogenic effect of vagal stimulation. Immunoblotting revealed that the MI-SS group showed a marked reduction in the amount of phosphorylated connexin43 (Cx43), whereas the MI-VS group showed only a slight reduction compared with the sham operation and sham stimulation group (37 +/- 20% versus 79 +/- 18%). Immunohistochemistry confirmed that the MI-induced loss of Cx43 from intercellular junctions was prevented by vagal stimulation. In addition, studies with rat primary-cultured cardiomyocytes demonstrated that acetylcholine effectively prevented the hypoxia-induced loss of phosphorylated Cx43 and ameliorated the loss of cell-to-cell communication as determined by Lucifer Yellow dye transfer assay, which supports the in vivo results.
Conclusions - Vagal nerve stimulation exerts antiarrhythmogenic effects accompanied by prevention of the loss of phosphorylated Cx43 during acute MI and thus plays a critical role in improving ischemia-induced electrical instability.

DOI： 10.1161/CIRCULATIONAHA.104.525493

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Electrical stimulation of the vagal efferent nerve improves the survival of myocardial infarcted rats. However, the mechanism for this beneficial effect is unclear. We investigated the effect of acetylcholine (ACh) on hypoxia-inducible factor (HIF)-1 alpha using rat cardiomyocytes under normoxia and hypoxia. ACh posttranslationally regulated HIF-1 alpha and increased its protein level under normoxia. ACh increased Akt phosphorylation, and wortmannin or atropine blocked this effect. Hypoxia-induced caspase-3 activation and mitochondrial membrane potential collapse were prevented by ACh. Dominant-negative HIF-1 alpha inhibited the cell protective effect of ACh. In acute myocardial ischemia, vagal nerve stimulation increased HIF-1 alpha expression and reduced the infarct size. These results suggest that ACh and vagal stimulation protect cardiomyocytes; through the PI3K/Akt/HIF-1 alpha pathway. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.02.065

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The dielectric properties (conductivity, kappa and relative permittivity, epsilon) of excised rat lung are modified by lung air and water content. The measurements of these quantities were made over the frequency range of 10 kHz to 100 MHz with an open-ended coaxial probe. The following relationships were analyzed in an oleic acid-induced pulmonary edema model using 18 animals: the spectra Of kappa, epsilon and the loss tangent as a function of lung air and water content. Secondly, an isolated-perfused lung system was produced to induce a gradual increase in lung water. The time course Of kappa, epsilon and the loss tangent for one excised lung was analyzed. The principal findings were: (i) a decrease in kappa and epsilon with increasing air content, (ii) an increase in kappa and epsilon with increasing water content, and (iii) a good correlation between lung water content and maximum loss tangent that was insensitive to changes in air content.
We conclude that this technique could provide a quantitative assessment of lung water during pulmonary edema formation. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.resp.2004.08.008

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Endothelin-1 (ET-1) is known as an aggravating factor of the failing cardiomyocytes and, therefore, a therapeutic method is indispensable to decrease cardiac ET-1 expression. To study the mechanisms of how cardiac ET-1 gene expression can be modified, we investigated the effect of electrical stimulation against cardiomyocytes. Considering the physiology of cardiomyocytes, in vitro cultured cardiomyocytes demonstrate distinctive features from in vivo cardiomyocytes (i.e. the absence of a stretch along with electrical stimulation). In this study, we especially focused on the effect of electrical stimulation. The electrical stimulation reduced the gene expression of ET-1 mRNA in rat primary cultured cardiomyocytes. Furthermore, this effect on the transcriptional modification of ET-1 was also identified in H9c2 cells. Luciferase activity using H9c2 cells was decreased by electrical stimulation in the early phase, suggesting that the attenuation of the ET-1 gene transcription by electrical stimulation should be due to a transcriptional repression. To further investigate a trigger signal involved in the transcriptional repression, phosphorylation of 5'-AMP-activated protein kinase (AMPK) was evaluated. It was revealed that AMPK was phosphorylated in the early phase of electrical stimulation of H9c2 cells as well as in rat primary cultured cardiomyocytes, and that AMPK phosphorylation was followed by ET-1 transcriptional repression, suggesting that electrical stimulation directly regulates AMPK. This study suggests that AMPK activation in cardiomyocytes plays a crucial role in the transcriptional repression of ET-1.

DOI： 10.1097/01.fjc.0000166318.91623.f9

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The neural mechanisms of the thermoregulatory control of core and skin temperatures in response to heat and cold stresses have been well clarified. However, it has been unclear whether baroreceptor reflexes are involved in the control of core and skin temperatures. To investigate how the arterial baroreceptor reflex modulates the body temperatures, we examined the effect of pressure changes of carotid sinus baroreceptors on core and skin temperatures in halothane-anesthetized rats. To open the barore-flex loop and control arterial baroreceptor pressure (BRP), we cut vagal and aortic depressor nerves and isolated carotid sinuses. We sequentially altered BRP in 20-mmHg increments from 60 to 180 mmHg and then in 20-mmHg decrements from 180 to 60 mmHg while measuring systemic arterial pressure (SAP), heart rate (HR), and core blood temperature (T-core) at the aortic arch and skin temperature (T-skin) at the tail. In response to the incremental change in BRP by 120 mmHg, SAP, HR, and T-core fell by 90.3+/-5.1 mmHg, 60.3+/-10.5 beats min(-1), and 0.18+/-0.01degreesC, respectively. Tskin rose by 0.84+/-0.10degreesC. The maximum rate of change per unit BRP change was -2.1+/-0.2 for SAP, -1.5+/-0.4 beats min(-1) mmHg(-1) for HR, -0.003+/-0.001degreesC mmHg(-1) for Tcore, and 0.011+/-0.002degreesC mmHg(-1) for T-skin. After the administration of hexamethonium or bretylium, these baroreflexogenic responses were completely abolished. We concluded that Tcore and Tskin are modulated by the arterial baroreceptor reflex.

DOI： 10.2170/jjphysiol.53.461

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Cochlea endolymph, produced by the stria vascularis, is essential for normal inner ear function. Abnormal endolymphatic volumes correlate closely with pathological conditions such as Meniere's disease. The critical roles played by aquaporins, which facilitate osmotic movement of water molecules, are known in a variety of tissues. We investigated the expression of aquaporin-1 (AQP1) in the rat inner ear using reverse transcription polymerase chain reaction and immunohistochemical methods. We obtained novel data showing that not just AQP1 mRNA but also AQP1 protein is expressed in the stria vascularis, in addition to other data confirming previous reports. AQP1 immuno reactivity localized to the intermediate cells in the stria vascularis. The above finding suggests that AQP1 may play a role in the water distribution associated with vigorous ion transport in the stria vascularis since the intermediate part of the stria vascularis contains both intermediate cells and the basolateral parts of marginal cells, both of which express ion transporters abundantly. (C) 2003 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-5955(03)00131-X

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The cochlear stria vascularis produces the endolymph and generates the endocochlear DC potential, two indispensable ingredients of an auditory transduction process. The marginal cell, one of the several cell types constituting the stria vascularis, is called 'the dark cell' on the basis of its appearance by transmission electron microscopy (TEM). To clarify whether this commonly observed 'dark appearance' is a normal characteristic of marginal cells, as conjectured in the literature, or an experimental artifact, we developed an in vivo fixation method for minimizing ischemic tissue damages. While under sustained systemic circulation with oxygenated blood, the stria vascularis of gerbils was chemically fixed by perilymphatic perfusion with a fixative, and the stria vascularis was observed by TEM. In contrast to a number of previous reports, the cytoplasm of marginal cells was not dark, and quantitative analysis showed that the difference between the cytoplasmic electron density of marginal cells and that of intermediate cells (another type of strial cells) was not statistically significant. For comparison, the gerbils were allowed to undergo 3 min of ischemia following decapitation. Under these conditions, marginal cells showed typical 'dark appearance', as reported previously, and their cytoplasmic electron density was 1.7 times higher than that of the intermediate cells. In addition, the volume of mitochondria in marginal cells undergoing 3 min of ischemia was higher than that fixed in vivo. We therefore conclude that the widely recognized 'dark cell' appearance of marginal cells following conventional fixation procedures reflects cell injury due to ischemia, which is inherent in the standard fixation procedures, but can be avoided by our fixation protocol here introduced.

DOI： 10.1007/s00441-002-0597-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In an earlier publication (Takeuchi et al., Biophys. J. 79 (2000) 2572 2582), we proposed that K channels in intermediate cells within the stria vascularis may play an essential role in the generation or the endocochlear potential (EP). and we presented an extended version of the five-compartment model of the stria vascularis. In search of further evidence supporting the five-compartment model, we studied the effects of Cs+ added to the perilymph on guinea pig EP, Cs+ is known as a competitive K+ channel blocker. Both the scala tympani and the scala vestibuli of four cochlear turns were perfused at a flow rate of 10 mul/min, and the EP was recorded from the second cochlear turn. Cs- at 30 mM caused a biphasic change in the EP the EP increased transiently from a control level of 89.6 mV to 94.8 mV within 10 min, and then decreased to a. steady level of 24.5 mV within the next 40 min. We propose that the initial transient increase in the EP results from Cs+-mediated blockade of K+ conductance in the basolateral membrane of hair cells. and that the subsequent EP decrease is due to effects of Cs+ on the stria vascularis. We believe that Cs- in the perilymph is able to access the stria vascularis by being taken up by fibrocytes in the spiral ligament and then being transported to intermediate cells because it is known that Cs+ is taken up via Na+, K+-ATPase and that gap junctions connect fibrocytes in the spiral ligament to basal cells and basal cells to intermediate cells. To clarify the effect of intracellular Cs+ on the electrophysiological properties of intermediate cells. these cells were dissociated from guinea pigs and Studied by the whole-cell patch-clamp method. intracellular Cs+ depolarized intermediate cells in a dose-dependent manner. In addition, efflux of Cs+ from the intermediate cell was much less than the efflux of K+. Thus, Cs+ may accumulate in the intermediate cell, which depolarizes the cell, which in turn decreases the EP. We conclude that the five-compartment model of the stria vascularis can explain the EP decrease caused by Cs+ in the perilymph. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-5955(01)00412-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS AS  

Objective-Loop diuretics such as bumetanide and furosemide cause an acute enlargement of the intrastrial space of the stria vascularis, with an associated decline in the endocochlear DC potential (EP). The aim of this study was to determine the role played by the Na+-K+-ATPase in the bumetanide-induced enlargement of the intrastrial space, and to examine the importance of the balance between the activities of the Na+-K+-2Cl(-) cotransporter and the Na+-K+-ATPase to the physiological function of the stria vascularis.
Material and methods-Albino guinea pigs were used in experiments involving perilymphatic perfusion, EP measurement and electron microscopy. The effects of bumetanide on the stria vascularis were examined following inhibition of the Na+-K+-ATPase by ouabain. Ouabain was administered to the perfusate and, when the EP reached 0 mV, both ouabain and bumetanide were administered.
Results-Although there was no enlargement of the intrastrial space, vacuoles were apparent in marginal cells. The vacuolar change in marginal cells was similar to that caused by ouabain alone.
Conclusion-This study indicates that the enlargement of the intrastrial space requires not only the blockade of the Na+-K+-2Cl(-) cotransporter but also normal activity of the Na+-K+-ATPase, and suggests that the bumetanide-induced enlargement of the intrastrial space resulted from the imbalance between the activities of the Na+-K+-2Cl(-) cotransporter and the Na+-K+-ATPase.

DOI： 10.1080/003655402/000028051

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Language：Japanese   Publisher：THE JAPAN OTOLOGICAL SOCIETY  

The lateral wall of the cochlear duct dissected from gerbils or rats was studied by confocal laser microscopy. Specific structures in tissue preparations were stained with fluorescent dyes to clarify the following points: i) developmental changes in the capillary network in the lateral wall of the cochlear duct, ii) intercellular connection by gap junctions, iii) distribution of elongated cells connecting capillaries, and iv) three dimensional distribution of intermediate cells in the stria vascularis. These studies revealed structural characteristics related to specific functions of the stria vascularis.

DOI： 10.11289/otoljpn1991.11.84

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Structural relationships between intermediate cells and capillaries in the stria vascularis of gerbils were examined by confocal laser microscopy and electron microscopy. Immunustaining for an inward rectifier K+ channel (Kir4.1), which was localized to intermediate cells, was used to determine the three-dimensional distribution of intermediate cells. These cells constituted a honeycomb-like network, and their dendritic processes surrounded not only capillaries but also the basolateral surface of epithelial marginal cells. On the basis of the above finding and the large K+ conductance in intermediate cells, M e propose that the network composed of intermediate cells has a spatial K+ buffering function. Transmission electron microscopy revealed the absence of the basal lamina in some regions and the presence of a gap junction-like membrane association between intermediate cells and pericytes: and/or endothelial cells. This: result supported our previous finding that intermediate cells were dye-coupled with pericytes and endothelial cells. The presence of gap junctions between intermediate cells and pericytes and/or endothelial cells suggests that endothelial cells and pericytes may play roles other than forming a structural route for blood circulation. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-5955(01)00252-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOPHYSICAL SOCIETY  

The endocochlear DC potential (EP) is generated by the stria vascularis, and essential for the normal function of hair cells. Intermediate cells are melanocytes in the stria vascularis. To examine the contribution of the membrane potential of intermediate cells (E-m) to the EP, a comparison was made between the effects of K+ channel blockers on the E-m and those on the EP. The E-m of dissociated guinea pig intermediate cells was measured in the zero-current clamp mode of the whole-cell patch clamp configuration. The E-m changed by 55.1 mV per 10-fold changes in extracellular K+ concentration. Ba2+, Cs+, and quinine depressed the E-m in a dose-dependent manner, whereas tetraethylammonium at 30 mM and 4-aminopyridine at 10 mM had no effect. The reduction of the E-m by Ba2+ and Cs+ was enhanced by lowering the extracellular K+ concentration from 3.6 mM to 1.2 mM. To examine the effect of the K+ channel blockers on the EP, the EP of guinea pigs was maintained by vascular perfusion, and K+ channel blockers were administered to the artificial blood. Ba2+, Cs+ and quinine depressed the EP in a dose-dependent manner, whereas tetraethylammonium at 30 mM and 4-aminopyridine at 10 mM did not change the EP. A 10-fold increase in the K+ concentration in the artificial blood caused a minor decrease in the EP of only 10.6 mV. The changes in the EP were similar to those seen in the E-m obtained at the lower extracellular K+ concentration of 1.2 mM. On the basis of these results, we propose that the EP is critically dependent on the voltage jump across the plasma membrane of intermediate cells, and that K+ concentration in the intercellular space in the stria vascularis may be actively controlled at a concentration lower than the plasma level.

DOI： 10.1016/S0006-3495(00)76497-6

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

The cochlear stria vascularis is essential for the normal function of hair cells, mRNA encoding the CIC-K1 Cl- channel, previously thought to be found only in the kidney, was detected in epithelial marginal cells of the rat stria vascularis by single cell RT-PCR. When Cl- currents were recorded from rat marginal cells by the whole-cell patch clamp method, the steady-state currents showed weak outward rectification and an ion selectivity sequence of SCN- > Br- = Cl- > F- > NO3- > I- > gluconate(-). The Cl- currents were regulated by extracellular Ca2+ and pH. These characteristics resemble those reported for the currents recorded from Xenopus oocytes expressing CIC-K1 Cl- channels. These data together suggest that CIC-K1 Cl- channels may contribute to the whole-cell currents of marginal cells. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3940(00)01021-1

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The cochlear stria vascularis produces the positive endocochlear potential (EP) and the endolymph. Both the EP and the endolymph are essential for the physiological function of hair cells. The intermediate cell is one of several cell types constituting the stria vascularis. It is known that inward rectifier K+ channels can play a constitutive role in the determination of the resting membrane potential. Localization of a member of the inward rectifier K+ channel family, Kir4.1, in the stria vascularis of gerbils and rats was investigated by immunological methods. A polyclonal antibody specific to the C-terminus of the rat Kir4.1 channel was raised in rabbits. Immunostaining of dissociated cells revealed that the Kir4.1 channel was localized to the intermediate cell, but not to the epithelial marginal cell. Subcellular localization of the Kir4.1 channel to the plasma membrane of the intermediate cell was confirmed by immunoelectron microscopy. Immunostaining of whole-tissue preparations revealed a network-like structure composed of intermediate cells. It seems likely that the Kir4.1 channel mediates the inwardly rectifying K+ current in the intermediate cell as shown previously by electrophysiological methods, and that this channel plays key roles in the production of the EP and K+ transport in the stria vascularis.

DOI： 10.1007/s004419900066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

A voltage-dependent outward KC (Kv) current in the intermediate cell (melanocyte) of the cochlear stria vascularis was studied using the whole cell patch-clamp technique. The Ky current had an activation threshold voltage of approximately -80 mV, and 50% activation was observed at -42.6 mV. The time courses of activation and inactivation were well fitted by two exponential functions: the time constants at 0 mV were 7.9 and 58.8 ms for activation and 0.6 and 4.3 s for inactivation. The half-maximal activation time was 13.8 ms at 0 mV. Inactivation of the current was incomplete even after a prolonged depolarization of 10 s. This current was independent of intracellular Ca2+. Quinine, verapamil, Ba2+, and tetraethylammonium inhibited the current in a dose-dependent manner, but 4-aminopyridine was ineffective at 50 mM. We conclude that the Ky conductance in the intermediate cell may stabilize the membrane potential, which is thought to be closely related to the endocochlear potential, and may provide an additional route for K+ secretion into the intercellular space.

DOI： 10.1152/ajpcell.1999.277.1.C91

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Bridging structures between discrete capillaries in the stria vascularis of the cochlea were studied morphologically in gerbils and rats. Serial thin sections for transmission electron microscopy revealed (1) that elongated cells surrounded by the basal lamina provided the structural basis for the bridging structure, (2) that the basal lamina surrounding the elongated cell extended to the basal lamina around the capillary endothelial cell, (3) that the electron density of the cytoplasm was similar to that of the pericytes around the capillaries, and (4) that the cell was attached to the capillaries at both ends only. Visualization of the basal lamina by immunofluorescent methods revealed (1) that capillaries were often bent at the site of attachment of the bridging cell, (2) that the bridging cell bifurcated occasionally, and (3) that the density of the bridging cell was much higher in the stria vascularis than in the underlying spiral ligament. Filamentous actin visualized by fluorescent phalloidin was not apparent in the bridging cell. We propose that the bridging cell provides mechanical strength to the tortuous capillary network in the stria vascularis and participates in the specific function of the stria vascularis in cooperation with other types of cells.

DOI： 10.1007/s004410051327

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The development of the capillary network in the stria vascularis and in the underlying spiral ligament of gerbils was systematically and quantitatively investigated by conventional electron microscopy and confocal laser microscopy in association with vascular labeling with fluorescent gelatin. The developmental changes of capillaries in the lateral wall were observed as the following series of events. (i) At 0 days after birth (DAB) capillaries already existed in the spiral ligament as a network. (ii) At 3-9 DAB the capillary network developed into two layers starting from the scala vestibuli side to the scala tympani side; one layer was located in the stria and the other in the spiral ligament. (iii) At 9 DAB capillaries in the stria became separated from the spiral ligament, and the capillary network consisting of a two-layered structure was complete. (iv) Total capillary length and capillary density in the lateral wall increased until 9 DAB and leveled off thereafter, but changes in the relative position of capillaries in the stria toward the luminal surface of marginal cells continued until 31 DAB. On the basis of the above observations, we propose two possible mechanisms underlying the vascular development in the lateral wall: (i) the formation of new vasculature (angiogenesis), and (ii) changes in the position of cellular components relative to capillaries in association with the differentiation and maturation of marginal cells and intermediate cells. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-5955(98)00109-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Intercellular connections via gap junctions in the stria vascularis, which constitutes the lateral wall of the cochlear duct, were investigated by the Lucifer yellow microinjection method with the aid of a confocal laser microscope. The dye injected into an intermediate cell (melanocyte) diffused into capillary endothelial cells and pericytes as well as other intermediate cells, basal cells, and fibrocytes in the spiral ligament; whereas the dye injected into a marginal cell (epithelial cell) was confined to the injected cell. The observation of dye-coupling between intermediate cells and endothelial cells and pericytes makes likely the possibility that these cells work together to play a role in the specific function of the stria vascularis (i.e., production of the positive endocochlear potential and the endolymph) and adds endothelial cells and pericytes to the current "two-cell model" of the stria vascularis.

DOI： 10.1007/s004410051118

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

The stria vascularis in the cochlea generates the endocochlear potential (EP) and secretes K+-rich endolymph; both are indispensable for normal sound transduction by hair cells. K+ conductance in the intermediate cell, one of the several types of cells constituting the stria vascularis, was investigated by the whole-cell patch-clamp technique. Inwardly-rectifying K+ (K-ir) currents were the major currents observed. The currents were inhibited dose-dependently by Ba2+, quinine, verapamil and Cs+, but not by tetraethylammonium (20 mM), 4-aminopyridine (5 mM) or Cd2+ (1 mM). The similarity between the effect of inhibitors on K-ir currents and on the EP (Takeuchi et al., Hearing Res., 101 (1996) 181-185) suggests a direct contribution of the K-ir conductance to the generation of the EP. (C) 1998 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0304-3940(98)00318-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Strial marginal cells are known to take up and metabolize glucose as their main source of metabolic energy. The membrane transport mechanisms for glucose uptake into strial marginal cells, however, are largely unknown. Two types of glucose transporters in mammalian cells have been described, the facilitated glucose transporter GLUT and the sodium/glucose cotransporter SGLT. The goal of the present study was to determine which of these represent the main glucose uptake mechanism in strial marginal cells. Glucose uptake into strial marginal cells was assessed by monitoring the cellular concentration of the reduced form of nicotinamide adenine dinucleotide (NADH) fluorometrically. The relation between the autofluorescence from marginal cells and cellular metabolism was verified as follows. The autofluorescence (excitation: 340 nm, emission: 450-490 nm) decreased when oxidative phosphorylation in the mitochondria was uncoupled with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and increased when cytochrome oxidase was inhibited with cyanide. These effects indicate that the autofluorescence is dependent on the mitochondrial metabolic state, and more specifically on the level of NADH in mitochondria. Glucose removal from the bath solution elicited a 39% decrease in the autofluorescence intensity within 5 min. Similarly, cytochalasin B (10 mu M) reduced the fluorescence intensity by 34% in 5 min. In contrast, neither phlorizin (0.1 mM) nor Na+ removal from the bath solution caused any appreciable change in the autofluorescence intensity. These results indicate that glucose depletion affects the metabolic state of the marginal cell within a few minutes, and that marginal cells take up glucose via GLUT, but not via SGLT. Since the excitation and emission wavelengths of several fluorescent dyes used in physiological studies (e.g., Fura-2 and SBFI) are similar to those of NADH, possible effects of autofluorescence on recording signals should always be taken into account when these dyes are utilized. (C) 1997 Elsevier Science B.V.

DOI： 10.1016/S0378-5955(97)00157-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Marginal cells constitute the endolymph-facing epithelium responsible for the secretion of endolymph by the stria vascularis in the inner ear. We have studied the possible involvement of Cl- conductance and Na+-K+-Cl- cotransport in the mechanism of changes in cell volume upon isotonic Cl- depletion/restoration. Changes in cell volume were estimated from video-microscopic images with the aid of an image processor. Marginal cells shrank to similar to 80% of their original volume in 30 s and to 65-70% in 90 s upon total replacement of [Cl](0) (similar to 150 mM) by gluconate(-), and the original volume of the shrunken cells was restored within 2 min after restoration of Cl-. The order of potency of anions to induce isotonic shrinkage was gluconate(-) > I- > F- > Br-. The cell shrinkage caused by Cl- depletion was partially inhibited by 5-Nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB, 0.2 mM), but not by either 4-acetamido-4'-isothiocyanato-stilbene-2,2'disulfonic acid (SITS, 0.5 mM), bumetanide (10 mu M) or ouabain (1 mM). The cell shrinkage caused by a reduction of [Cl](0) from similar to 150 mM to 7.5 mM was not affected by [K](0) in the range of 3.6 mM to 72 mM. These results suggest that the main efflux pathway(s) responsible for the 'Cl removal'-induced shrinkage depends on volume-correlated Cl- conductance (Takeuchi and Irimajiri, J. Membrane Biol. 150, 47-62, 1996) and that this pathway(s) is essentially independent of the Na+-K+-Cl- cotransporter, the Na+,K+-ATPase, and the K+-Cl- cotransporter. With regard to volume recovery after isotonic shrinkage, its critical dependence on the simultaneous presence of Na+, K+ and Cl- in the bath and its substantial inhibition by bumetanide (10 mu M) both indicate a major role for Na+-K+-Cl- cotransport. The strong influence on cell volume of solute fluxes working through the Cl- channel and the Na+-K+-Cl- cotransporter implies an essential role for these pathways in the ion transport mechanism(s) of the marginal cell.

DOI： 10.1016/S0378-5955(97)00134-2

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In search of a method for detecting rouleaux formation in vitro, we studied the dielectric behavior of human blood under both agitated and stationary conditions. Among the parameters examined, relative permittivity ('dielectric constant') at 50-100 kHz was found to be a suitable measure of rouleaux growth, which has been difficult to quantify through conventional optical approaches. The electrical method presented here appears applicable to the kinetic analysis of rouleaux formation in undiluted whole blood.

DOI： 10.1016/0304-4165(96)00048-7

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The basolateral membrane of isolated strial marginal cells has been probed for conductive pathways by the patch-damp technique. Two types of voltage-insensitive channels were identified in both cell-attached and excised patches. Of these, frequently (69% of excised patches) observed was a Ca2+-activated nonselective cation channel having a unit conductance of 24.9 +/- 0.5 pS (N = 16). Other characteristics of this type in excised patches include: 1) linear I-V relations with 150 mM K+ (pipette)/150 mM Na+ (bath), 2) a permeability sequence of NH4+ > Na+ = K+ = Rb+ > Li+, 3) a flickering block by quinine or quinidine (both 1 mM), and 3) a dose dependent block of its activity by ADP or ATP (IC50,ATP/IC50,ADP = 20-35), both from the cytosolic side. Channels with similar characteristics were found in the apical membrane of the same cell; however, the basolateral channels were 2-4 times more densely distributed than the apical counterparts. Also frequently (57%) detected was a Cl- channel of 80.0 +/- 0.5 pS (N = 6), whose activity was Ca2+ independent. Additionally, this Cl- channel had: 1) linear I-V relations with symmetric Cl-, 2) a permeability sequence of Cl- > Br- > I- greater than or equal to NO3- greater than or equal to gluconate(-), and 3) a complete and reversible block by 1 mM diphenylamine-2-carboxylate. In contrast to the apical Cl- channels, the basolateral ones had a much higher density (57% vs. < 1%) as well as a higher unit conductance (80 pS vs. 50 pS) than the apical counterpart. The relative abundance of these two types as the major conductive pathways for Na+, K+, and Cl- in the basolateral region must be taken into account when addressing the role of strial marginal cells in generating the positive endocochlear potential. The Cl- channel may facilitate Cl- distribution across the basolateral membrane.

DOI： 10.1016/0378-5955(94)00191-R

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

The heliozoan Echinosphaerium feeds on various kinds of small protozoans by slow retraction or rapid contraction of microtubule-containing axopodia. The axonemal microtubules showed an intense positive birefringence that was clearly visualized when the organism was observed under a highly sensitive polarization microscope. In order to know if the axopodial contraction involves rapid disassembly of the cytoskeletal microtubules, dynamic changes of the axonemal birefringence were analyzed here during the process of rapid axopodial contraction by video microscopy with an image digitizing system. The number of microtubules in an axopodium was estimated from its birefringence by applying dielectric theories for shell-covered ellipsoids in concentrated suspensions. Quantitative analysis showed that the fractional volume occupied by the microtubules increased at the proximal region of the axopodium concomitantly with the axopodial contraction, and it was maintained for at least 10 s after the contraction. Electron microscopic observations showed that two bundles of axonemal microtubules crossed each other in the proximal part of a contracted axopodium. These results indicate that, during rapid axopodial contraction, the distal portion of the axonemal microtubules moves as a bundle into the axopodial base without a distinct disassembly of the microtubules themselves.

DOI： 10.1016/S0932-4739(11)80215-4

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Language：Japanese   Publishing type：Research paper (scientific journal)  

To correlate changes in the passive electrical properties of the rabbit cornea with quantitative grading of corneal injuries induced by topical application of 70% ethanol, we measured ocular tissue impedances using a surface electrode over the range of 104 ~ 108 Hz and followed their temporal changes for up to 17 days. Dielectric measurements on control eyes yielded a broad dispersion curve, which, in loss tangent terms, could be decomposed into two components: dispersion 1 on the low-frequency side and dispersion 2 on the high-frequency side. By defining the peak value of the total dispersion as P(t) and those of subdispersions 1 and 2 P1 and P2, the ratios, P1/P(t) and P2/P(t), were found to serve as useful indices. Upon appearance of corneal erosion due to ethanol, P1/P(t) markedly increased and returned to the control level with re-epithelialization of the cornea, and the time course of P2/P(t) showed a mirror image to that of P1/P(t). Both ratios correlated well with the erosion area determined photographically. These results indicate that dielectric spectroscopy is applicable to the assessment of the extent and severity of corneal injury.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：VCH PUBLISHERS INC  

The large heliozoan Echinosphaerium extends a number of needle-like axopodia by which it captures food organisms. Every axopodium contains a bundle of several hundreds of axonemal microtubules as a cytoskeletal element. When the tip of a poly-L-lysine-coated glass micro-needle came into contact with the distal part of an axopodium, a rapid axopodial contraction (2.6 mm/s) occurred with a concomitant bending of the needle toward the cell body. In this report, we measured the force of the axopodial contraction by utilizing the relation between force and bending displacement of the micro-needle, and examined a possibility that the axopodial contraction is ascribed to the axopodial tension (surface tension and/or cytoplasmic elasticity) that is developed as a result of microtubule degradation. The force of the axopodial contraction was estimated in the order of 10(-9) N. Treatment with 10 mM colchicine induced disassembly of the axopodial microtubules and a resulting slow retraction of the axopodia (0.1 mum/s) occurred. The force of the slow retraction was also measured by the same procedure to estimate the axopodial tension, and was in the order of 10(-11) N. It was thus demonstrated that the motive force for axopodial contraction cannot be explained as an axopodial tension generated as a result of disassembly of the microtubules.

DOI： 10.1016/S0932-4739(11)80007-6

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Language：Japanese   Publisher：Hiroshima University  

Size distributions of intracellular organelles in an ultra-thin section are usually distorted when the section thickness is not negligible. A computer program is developed here to estimate true size distributions of spherical organelles from transmission electron micrographs of ultra-thin sections. The method is applied to determine size distributions of chromaffin granules isolated from bovine adrenal medullary cells as a basis for further physiological measurements.

DOI： 10.15027/622

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DOI： 10.1002/cm.970140214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC ELECTRON MICRO BUS CENTER ACAD SOC JAPAN  

DOI： 10.1093/oxfordjournals.jmicro.a050750

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Symposium, workshop panel (public)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Symposium, workshop panel (nominated)  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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