研究者詳細 - 安井　正人

論文 - 安井　正人

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Dynamic interactions of cations, water and lipids and influence on membrane fluidity

Kagawa Rina, Hirano Yoshinori, Taiji Makoto, Yasuoka Kenji, Yasui Masato

Journal of Membrane Science 435 130 - 136 2013年05月

ISSN 0376-7388

Cell membranes, which consist of a lipid bilayer, play a critical role in cellular bioactivities. The lipid bilayer interacts dynamically with water as well as ions. Membrane fluidity is lesser in ionic solutions than in pure water, although how ions reduce membrane fluidity has not yet been clarified. Here, we performed 4×100-ns molecular dynamics simulations of a POPE (1-palmitoyl-2-oleoyl-phosphatidyl-ethanolamine [18:1]) lipid bilayer in the presence or absence of ions (NaCl, KCl, or CaCl₂) and compared the systems to evaluate the influence of ions on the structural dynamics of membranes. Our simulation revealed that membrane fluidity (no ion-system>Ca-system>K-system>Na-system) did not correspond to the distribution or the direct binding of cations to lipids (Ca²⁺>Na⁺>K⁺). Interestingly, we found that the presence of cations induces dehydration of lipids. The number of cations in the interface region corresponded to the degree of lipid dehydration and the reduction in membrane fluidity (Na⁺>K⁺>Ca²⁺). We therefore propose that membrane fluidity is not affected by direct interactions between cations and lipids, but by the dehydration of lipid molecules in the presence of cations located adjacent to the membrane. © 2013 Elsevier B.V.

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Endfeet serve as diffusion-limited subcellular compartments in astrocytes

Nuriya Mutsuo, Yasui Masato

Journal of Neuroscience 33 （ 8 ） 3692 - 3698 2013年02月

ISSN 0270-6474

Astrocytes extend their processes to make contact with neurons and blood vessels and regulate important processes associated with the physiology/pathophysiology of the brain. Their elaborate morphology, with numerous fine processes, could allow them to perform complex signal transductions with distinct compartments or to function as a spatial buffer depending on the diffusion properties of their intracellular molecules. Apart from calcium ions, however, the diffusion dynamics of molecules within astrocytes are poorly understood. In this study, we applied two-photon uncaging and fluorescence recovery after photobleaching of fluorescent molecules to acute cortical brain slices from mice to investigate the diffusion dynamics of molecules within astrocytes. We found that diffusion was significantly more restricted at the endfeet than at trunks and distal ends of other processes. Slow diffusion dynamics at the endfeet resulted in a large population of molecules being retained in a small region for tens of seconds, creating subcellular compartments that were isolated from other regions. In contrast, diffusion was fast and free at other processes. The same patterns were observed with the diffusions of a higher molecular weight (10 kDa) molecule and 2-NBDG, a fluorescent analog of glucose. These findings suggest that molecular diffusion is not uniform across the intracellular environment and that subcellular compartments are present in astrocytes. Therefore, similar to neurons, the elaborate and specialized structures of astrocytes may enable them to perform complex computations by providing distinct information storage/processing capacity among processes. © 2013 the authors.

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Characterization of the role of a mechanosensitive channel in osmotic down shock adaptation in Synechocystis sp PCC 6803

Nanatani Kei, Shijuku Toshiaki, Akai Masaro, Yukutake Yoshinori, Yasui Masato, Hamamoto Shin, Onai Kiyoshi, Morishita Megumi, Ishiura Masahiro, Uozumi Nobuyuki

Channels 7 （ 4 ） 238 - 242 2013年

ISSN 1933-6950

Synechocystis sp strain PCC 6803 contains one gene encoding a putative large conductance mechanosensitive channel homolog [named SyMscL (slr0875)]. However, it is unclear whether SyMscL contributes to the adaptation to hypoosmotic stress in Synechocystis. Here we report the in vivo characteristics of SyMscL. SyMscL was mainly expressed in the plasma membrane of Synechocystis. Cell volume monitoring using stopped-flow spectrophotometry showed that ΔsymscL cells swelled more rapidly than wild-type cells under hypoosmotic stress conditions. Expression of symscL was under circadian control, and its peak corresponded to the beginning of subjective night. These results indicate that SyMscL functioned as one component of the osmotic homeostatic regulatory system of the cell coordinating the response of Synechocystis to daily metabolic osmotic fluctuations and environmental changes. © 2013 Landes Bioscience.

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Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4

Miyazaki Kaori, Abe Yoichiro, Iwanari Hiroko, Suzuki Yota, Kikuchi Takahiro, Ito Takashi, Kato Jungo, Kusano-Arai Osamu, Takahashi Toshiyuki, Nishiyama Shuhei, Ikeshima-Kataoka Hiroko, Tsuji Shoji, Arimitsu Takeshi, Kato Yasuhiro, Sakihama Toshiko, Toyama Yoshiaki, Fujihara Kazuo, Hamakubo Takao, Yasui Masato

Journal of Neuroimmunology 260 （ 1-2 ） 107 - 116 2013年

ISSN 0165-5728

Neuromyelitis optica is a demyelinating disease characterized by a disease-specific autoantibody designated as NMO-IgG that specifically recognizes aquaporin-4, and the binding of NMO-IgG to AQP4 causes the progress of the disease. Prevention of the binding of NMO-IgG, therefore, may alleviate the disease. Here we have developed monoclonal antibodies against AQP4 with a baculovirus display system in order to obtain high affinity monoclonal antibodies against the extracellular domains of AQP4. Our monoclonal antibodies can block the binding of NMO-IgG in spite of their heterogeneity. Taken together, we propose that our monoclonal antibodies can be applied in clinical therapy for NMO patients. © 2013 Elsevier B.V.

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Common force field thermodynamics of cholesterol

Giangreco Francesco, Yamamoto Eiji, Hirano Yoshinori, Hodoscek Milan, Knecht Volker, Di Giosia Matteo, Calvaresi Matteo, Zerbetto Francesco, Yasuoka Kenji, Narumi Tetsu, Yasui Masato, Höfinger Siegfried

The Scientific World Journal 2013 2013年

Four different force fields are examined for dynamic characteristics using cholesterol as a case study. The extent to which various types of internal degrees of freedom become thermodynamically relevant is evaluated by means of principal component analysis. More complex degrees of freedom (angle bending, dihedral rotations) show a trend towards force field independence. Moreover, charge assignments for membrane-embedded compounds are revealed to be critical with significant impact on biological reasoning. © 2013 Francesco Giangreco et al.

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Direct visualization and quantitative analysis of water diffusion in complex biological tissues using CARS microscopy

Yu Ying Chun, Sohma Yoshiro, Takimoto Shinichi, Miyauchi Takayuki, Yasui Masato

Scientific Reports 3 2013年

To date, it has not been possible to measure microscopic diffusive water movements in epithelia and in the interstitial space of complex tissues and organs. Diffusive water movements are essential for life because they convey physiologically important small molecules, e.g. nutrients and signaling ligands throughout the extracellular space of complex tissues. Here we report the development of a novel method for the direct observation and quantitative analysis of water diffusion dynamics in a biologically organized tissue using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Using a computer simulation model to analyze the CARS O-H bond vibration data during H 2 O/D 2 O exchange in a 3D epithelial cyst, we succeeded in measuring the diffusive water permeability of the individual luminal and basolateral water pathways and also their response to hormonal stimulation. Our technique will be applicable to the measurement of diffusive water movements in other structurally complex and medically important tissues and organs.

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Aquaporin AqpZ is involved in cell volume regulation and sensitivity to osmotic stress in Synechocystis sp. strain PCC 6803

Akai Masaro, Onai Kiyoshi, Morishita Megumi, Mino Hiroyuki, Shijuku Toshiaki, Maruyama Hisataka, Arai Fumihito, Itoh Shigeru, Hazama Akihiro, Checchetto Vanessa, Szabò Ildikò, Yukutake Yoshinori, Suematsu Makoto, Yasui Masato, Ishiura Masahiro, Uozumia Nobuyuki

Journal of Bacteriology 194 （ 24 ） 6828 - 6836 2012年10月

ISSN 0021-9193

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ΔaqpZ strain than the wild type. The ΔaqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ΔaqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity. © 2012, American Society for Microbiology.

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Structural features of aquaporin 4 supporting the formation of arrays and junctions in biomembranes

Höfinger Siegfried, Yamamoto Eiji, Hirano Yoshinori, Zerbetto Francesco, Narumi Tetsu, Yasuoka Kenji, Yasui Masato

Biochimica et Biophysica Acta - Biomembranes 1818 （ 9 ） 2234 - 2243 2012年09月

ISSN 0005-2736

A limited class of aquaporins has been described to form regular arrays and junctions in membranes. The biological significance of these structures, however, remains uncertain. Here we analyze the underlying physical principles with the help of a computational procedure that takes into account protein-protein as well as protein-membrane interactions. Experimentally observed array/junction structures are systematically (dis)assembled and major driving forces identified. Aquaporin 4 was found to be markedly different from the non-junction forming aquaporin 1. The environmental stabilization resulting from embedding into the biomembrane was identified as the main driving force. This highlights the role of protein-membrane interactions in aquaporin 4. Analysis of the type presented here can help to decipher the biological role of membrane arrays and junctions formed by aquaporin. © 2012 Elsevier B.V. All rights reserved.

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A phosphatidylinositol lipids system, lamellipodin, and Ena/VASP regulate dynamic morphology of multipolar migrating cells in the developing cerebral cortex

Yoshinaga Satoshi, Ohkubo Takahiro, Sasaki Shinji, Nuriya Mutsuo, Ogawa Yukino, Yasui Masato, Tabata Hidenori, Nakajima Kazunori

Journal of Neuroscience 32 （ 34 ） 11643 - 11656 2012年08月

ISSN 0270-6474

In the developing mammalian cerebral cortex, excitatory neurons are generated in the ventricular zone (VZ) and subventricular zone; these neurons migrate toward the pial surface. The neurons generated in theVZassume a multipolar morphology and remain in a narrow region called the multipolar cell accumulation zone (MAZ) for~24 h, in which they extend and retract multiple processes dynamically. They eventually extend an axon tangentially and begin radial migration using a migratory mode called locomotion. Despite the potential biological importance of the process movement of multipolar cells, the molecular mechanisms remain to be elucidated. Here, we observed that the processes of mouse multipolar cells were actin rich and morphologically resembled the filopodia and lamellipodia in growth cones; thus, we focused on the actin-remodeling proteins Lamellipodin (Lpd) and Ena/vasodilator-stimulated phosphoprotein (VASP). Lpd binds to phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P₂] and recruits Ena/VASP, which promotes the assembly of actin filaments, to the plasma membranes. In situ hybridization and immunohistochemistry revealed that Lpd is expressed in multipolar cells in the MAZ. The functional silencing of either Lpd or Ena/VASP decreased the number of primary processes. Immunostaining and a Förster resonance energy transfer analysis revealed the subcellular localization of PI(3,4)P₂ at the tips of the processes. A knockdown experiment and treatment with an inhibitor for Src homology 2-containing inositol phosphatase-2, a 5-phosphatase that produces PI(3,4)P₂ from phosphatidylinositol (3,4,5)-triphosphate, decreased the number of primary processes. Our observations suggest that PI(3,4)P₂, Lpd, and Ena/VASP are involved in the process movement of multipolar migrating cells. ©2012 the authors.

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Diffusive nature of xenon anesthetic changes properties of a lipid bilayer

Yamamoto Eiji, Akimoto Takuma, Shimizu Hiroyuki, Hirano Yoshinori, Yasui Masato, Yasuoka Kenji

Journal of Physical Chemistry B 116 （ 30 ） 8989 - 8995 2012年08月

ISSN 1520-6106

Effects of general anesthesia can be controllable by the ambient pressure. We perform molecular dynamics simulations for a 1-palmitoyl-2-oleoyl phosphatidylethanolamine lipid bilayer with or without xenon molecules by changing the pressure to elucidate the mechanism of the pressure reversal of general anesthesia. According to the diffusive nature of xenon molecules in the lipid bilayer, a decrease in the orientational order of the lipid tails, an increase in the area and volume per lipid molecule, and an increase in the diffusivity of lipid molecules are observed. We show that the properties of the lipid bilayer with xenon molecules at high pressure come close to those without xenon molecules at 0.1 MPa. Furthermore, we find that xenon molecules are concentrated in the middle of the lipid bilayer at high pressures by the pushing effect and that the diffusivity of xenon molecules is suppressed. These results suggest that the pressure reversal originates from a jamming and suppression of the diffusivity of xenon molecules in lipid bilayers. © 2012 American Chemical Society.

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Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase

Hiyoshi H., Abdelhady S., Segerström L., Sveinbjörnsson B., Nuriya M., Lundgren T. K., Desfrere L., Miyakawa A., Yasui M., Kogner P., Johnsen J. I., Andäng M., Uhlén P.

British Journal of Cancer 106 （ 11 ） 1807 - 1815 2012年05月

ISSN 0007-0920

Background: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. Methods: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. Results: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21 ^Waf1/Cip1 and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. Conclusion: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies. © 2012 Cancer Research UK.

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[Role of aquaporin, water channel in the brain].

Yasui Masato

Nihon yakurigaku zasshi. Folia pharmacologica Japonica 139 （ 5 ） 230 2012年05月

ISSN 0015-5691

An astrocyte-specific enhacer of the aquaporin-4 gene functions through a consensus sequence of POU transcription factors in concert with multiple upstream elements

Abe Yoichiro, Ikeshima-Kataoka Hiroko, Goda Wakami, Niikura Takako, Yasui Masato

Journal of Neurochemistry 120 （ 6 ） 899 - 912 2012年03月

ISSN 0022-3042

Aquaporin-4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin-4 gene, respectively. To identify cis-acting elements responsible for the astrocyte-specific transcription of M1 mRNA, the promoter activity of the 5′-flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte-specific activity enhancing transcription from the M1 promoter was observed within ∼2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286-bp region were found to function as a novel astrocyte-specific enhancer. Among the five elements, a consensus sequence of Pit-1/Oct/Unc-86 (POU) transcription factors was indispensable to the astrocyte-specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

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Pharmaceutical implications of curcumin in the development of drugs for cf pharmacotherapy

Sohma Yoshiro, Yu Ying chun, Miki Haruna, Nakamura Yumi, Hanyuda Akiko, Lin Hui Yi, Yasui Masato, Bompadre Silvia, Hwang Tzyh Chang

223 - 234 2012年02月

The Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel plays an essential role in salt and water transport across epithelia and mutations of CFTR causing its dysfunction result in the genetic disease cystic fibrosis (CF). Various mutations induce CFTR channel dysfunction and various pharmacological approaches are currently being explored. The G551D mutation in CFTR is a common cause of CF, characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Recently we investigated the potentiation effect of curcumin, especially its combined effect with a flavonoid genistein, on G551D-CFTR activity using the patch-clamp technique. Curcumin potentiated G551D-CFTR less than genistein at their maximally effective concentrations. However, curcumin showed an additive effect with genistein and, more importantly, a significant synergistic effect with genistein in a low concentration range. This does not only suggest that multiple mechanisms are involved in action of the CFTR potentiators, but also pose pharmaceutical implications of curcumin in the development of drugs for CF pharmacotherpy. © 2012 Nova Science Publishers, Inc. All rights reserved.

Increased expression of aquaporin-4 with methylmercury exposure in the brain of the common marmoset

Yamamoto Megumi, Takeya Motohiro, Ikeshima-Kataoka Hiroko, Yasui Masato, Kawasaki Yasuaki, Shiraishi Mitsuya, Majima Eiji, Shiraishi Seiji, Uezono Yasuhito, Sasaki Masanori, Eto Komyo

Journal of Toxicological Sciences 37 （ 4 ） 749 - 763 2012年

ISSN 1880-3989

The relationship between methylmercury (MeHg) exposure and aquaporin (AQP) expression in the brain is currently unknown. To investigate this, we used a common marmoset model of acute MeHg exposure to examine AQP1, AQP4 and AQP11 gene expression. MeHg (1.5 mg Hg/kg/day p.o.) was given to three marmosets for 14 days, followed by 14 days without. All treated marmosets showed slight akinesia before sacrifice. In the frontal lobe, occipital lobe and cerebellum, total mercury concentrations following MeHg administration were 26.7, 31.4, and 22.6 μg/g, respectively. Slight apoptosis was observed in the occipital lobe. Immunohistochemistry showed increased expression of glial fibrillary acidic protein, its mRNA and Iba1 with MeHg, indicating that neuronal injury activated astrocytes and microglia. There was no significant difference between control and MeHg-administered groups in AQP1 protein or AQP11 mRNA in the frontal lobe, occipital lobe or cerebellum. The ratio of AQP4 mRNA expression in MeHg-administered marmosets to the mean AQR4 expression in the controls (n = 3) were 1.3, 1.5 and 1.2, 1.7, 1.9 and 1.5, and 1.5, 1.6 and 1.2 for the frontal lobe, occipital lobe and cerebellum, respectively. Western blotting showed significantly increased AQP4 protein in the occipital lobe and cerebellum with MeHg administration, but no obvious up-regulation in the frontal lobe. Immunofluorescence analysis with double staining revealed low AQP4 expression in the cell body of reactive astrocytes in the MeHg-administered group. These results indicate that AQP4 expression might be stimulated by MeHg exposure in astrocytes in the occipital lobe and cerebellum, suggesting a role for AQP4 in MeHg neurotoxicity via astrocyte dysfunction.

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Non-Gaussian fluctuations resulting from power-law trapping in a lipid bilayer

Akimoto Takuma, Yamamoto Eiji, Yasuoka Kenji, Hirano Yoshinori, Yasui Masato

Physical Review Letters 107 （ 17 ） 2011年10月

ISSN 0031-9007

Anomalous diffusion in lipid bilayers is usually attributed to viscoelastic behavior. We compute the scaling exponent of relative fluctuations of the time-averaged mean square displacement in a lipid bilayer, by using a molecular dynamics simulation. According to the continuous time random walk theory, this exponent indicates non-Gaussian behavior caused by a power-law trapping time. Our results provide the first evidence that a lipid bilayer has not only viscoelastic properties but also trapping times distributed according to a power law. © 2011 American Physical Society.

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Plasma membrane aquaporin AqpZ protein is essential for glucose metabolism during photomixotrophic growth of Synechocystis sp. PCC 6803

Akai Masaro, Onai Kiyoshi, Kusano Miyako, Sato Mayuko, Redestig Henning, Toyooka Kiminori, Morishita Megumi, Miyake Hiroshi, Hazama Akihiro, Checchetto Vanessa, Szabò Ildikò, Matsuoka Ken, Saito Kazuki, Yasui Masato, Ishiura Masahiro, Uozumi Nobuyuki

Journal of Biological Chemistry 286 （ 28 ） 25224 - 25235 2011年07月

ISSN 0021-9258

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity atpHvalues ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Curcumin and genistein additively potentiate G551D-CFTR

Yu Ying Chun, Miki Haruna, Nakamura Yumi, Hanyuda Akiko, Matsuzaki Yohei, Abe Yoichiro, Yasui Masato, Tanaka Kazuhiko, Hwang Tzyh Chang, Bompadre Silvia G., Sohma Yoshiro

Journal of Cystic Fibrosis 10 （ 4 ） 243 - 252 2011年07月

ISSN 1569-1993

Background: The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. Methods: We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. Results: Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~. 50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. Conclusions: The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy. © 2011 European Cystic Fibrosis Society.

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Progesterone maintains amniotic tight junctions during midpregnancy in mice

Kobayashi Ken, Miwa Hideki, Yasui Masato

Molecular and Cellular Endocrinology 337 （ 1-2 ） 36 - 42 2011年04月

ISSN 0303-7207

The amniotic epithelium is in direct contact with the amniotic fluid and restricts fluid flux via the paracellular pathway by means of tight junctions (TJs). Several factors affect TJs to modulate the paracellular flux. Progesterone contributes to the antenatal formation and disappearance of TJs in uterine and mammary epithelial tissues. In this study, we investigated whether progesterone positively or negatively influences amniotic TJs. The administration of RU-486, a progesterone receptor (PR) antagonist, into pregnant mice adversely affects the localization and expression of claudin-3 and claudin-4 in the amniotic epithelium. RU-486 administration also increased the permeability of the amniotic membrane. In organ-cultured amniotic membranes, progesterone induced increases in claudin-3 and claudin-4 expression in a dose-dependent manner but did not influence their localization. PRs were also present in the amniotic epithelium during midpregnancy but they disappeared during late pregnancy. These results indicate that the progesterone/PR pathway maintains TJs in the amniotic epithelium during midpregnancy. © 2011 Elsevier Ireland Ltd.

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Apoptosis in mouse amniotic epithelium is induced by activated macrophages through the TNF receptor type 1/TNF pathway

Kobayashi Ken, Umezawa Kazuo, Yasui Masato

Biology of Reproduction 84 （ 2 ） 248 - 254 2011年02月

ISSN 0006-3363

The amniotic epithelium is in direct contact with the amniotic fluid and has tight junctions. The amniotic tight junctions function as a barrier to restrict fluid flux via the amniotic membrane during midpregnancy in the mouse. However, during late pregnancy, amniotic fluid volume significantly decreases in association with the disruption of amniotic tight junctions. The disruption of amniotic tight junctions is caused by apoptosis in the amniotic epithelium on Embryonic Day 17 (E17). In this study, we examine the molecular mechanisms underlying apoptosis of the amniotic epithelium of the mouse. We found that from E16, the number of activated macrophages that express high levels of NOS2 and tumor necrosis factor (TNF) increase in amniotic fluid. TNF receptor type 1 (TNFR1) was detectable from E16 onward. On E17, amniotic epithelial cells expressing TNFR1 became TUNEL positive, suggesting that TNF/TNFR1 signaling may initiate apoptosis. To further confirm the role of TNF/TNFR1 signaling, WP9QY, a TNFR1 antagonist, was injected into the amniotic cavity and was found to significantly reduce the numbers of apoptotic cells in the E17 amniotic epithelium. Furthermore, dehydroxymethylepoxyquinomicin, a specific nuclear factor-kappa B inhibitor, was found to inhibit TNF production in macrophages and amniotic apoptosis in vivo. Finally, we showed that injection of TNF into the amniotic cavity induces early onset of apoptosis. These results indicate that amniotic apoptosis is induced by the TNF pathway via TNFR1 expressed in the amniotic epithelial cells and that activation of macrophages may trigger amniotic apoptosis. © 2011 by the Society for the Study of Reproduction, Inc.

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