Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-VCH Verlag  

Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC20) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC20, actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4–5 fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC20s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone.

DOI： 10.1002/elps.201700394

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Salinity stress significantly reduces the root hydraulic conductivity (Lp(r)) of several plant species including barley (Hordeum vulgare). Here we characterized changes in the Lp(r) of barley plants in response to salinity/osmotic stress in detail using a pressure chamber. Salt-tolerant and intermediate barley cultivars, K305 and Haruna-nijyo, but not a salt-sensitive cultivar, I743, exhibited characteristic time-dependent Lp(r) changes induced by 100 mM NaCl. An identical response was evoked by isotonic sorbitol, indicating that this phenomenon was triggered by osmotic imbalances. Further examination of this mechanism using barley cv. Haruna-nijyo plants in combination with the use of various inhibitors suggested that various cellular processes such as protein phosphorylation/dephosphorylation and membrane internalization appear to be involved. Interestingly, the three above-mentioned barley cultivars did not exhibit a remarkable difference in root cell sap osmolality under hypertonic conditions, in contrast to the case of Lp(r). The possible biological significance of the regulation of Lp(r) in barley plants upon salinity/osmotic stress is discussed.

DOI： 10.1093/pcp/pcv013

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

The previously reported pressure-volume (PV) relationship in frog hearts shows that end-systolic PV relation (ESPVR) is load dependent, whereas ESPVR in canine hearts is load independent. To study intrinsic cardiac mechanics in detail, it is desirable to study mechanics in a single isolated cardiomyocyte that is free from interstitial connective tissue. Previous single cell mechanics studies used a pair of carbon fibers (CF) attached to the upper surface of opposite cell ends to stretch cells. These studies showed that end-systolic force-length (FL) relation (ESFLR) is load independent. However, the range of applicable mechanical load using the conventional technique is limited because of weak cell-CF attachment. Therefore, the behavior of ESFLR in single cells under physiologically possible conditions of greater load is not yet well known. To cover wider loading range, we contrived a new method to hold cell-ends more firmly using two pairs of CF attached to both upper and bottom surfaces of cells. The new method allowed stretching cells to 2.2 mu m or more in end-diastolic sarcomere length. ESFLR virtually behaves in a load independent manner only with end-diastolic sarcomere length less than 1.95 mu m. It exhibited clear load dependency with higher preload, especially with low afterload conditions. Instantaneous cellular elastance curves showed that decreasing afterload enhanced relaxation and slowed time to peak elastance, as previously reported. A simulation study of a mathematical model with detailed description of thin filament activation suggested that velocity dependent thin filament inactivation is crucial for the observed load dependent behaviors and previously reported afterload dependent change in Ca2+ transient shape. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pbiomolbio.2014.06.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2; 1, HvPIP2; 2, HvPIP2; 3 and HvPIP2; 5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2; 4 that is highly homologous to HvPIP2; 3 did not. The isoleucine residue at position 254 of HvPIP2; 3 was conserved in PIP2 aquaporins of barley, except HvPIP2; 4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2; 3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

DOI： 10.1093/pcp/pcu003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

OsPIP1;1 is one of the most abundant aquaporins in rice leaves and roots and is highly responsible to environmental stresses. However, its biochemical and physiological functions are still largely unknown. The oocyte assay data showed OsPIP1;1 had lower water channel activity in contrast to OsPIP2;1. EGFP and immunoelectron microscopy studies revealed OsPIP1;1 was predominantly localized in not only plasma membrane but also in some ER-like intracellular compartments in the cells. OsPIP1;1 exhibited low water channel activity in Xenopus oocytes but coexpression of OsPIP2;1 significantly enhanced its water permeability. Stop-flow assay indicated that 10His-OsPIP1;1-reconstituted proteoliposomes had significantly higher water permeability than the control liposomes. Overexpression of OsPIP1;1 greatly altered many physiological features of transgenic plants in a dosage-dependent manner. Moderate expression of OsPIP1;1 increased rice seed yield, salt resistance, root hydraulic conductivity, and seed germination rate. This work suggests OsPIP1;1 functions as an active water channel and plays important physiological roles. Published by Elsevier Masson SAS.

DOI： 10.1016/j.plaphy.2012.11.018

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Language：English   Publisher：Japanese Society for Medical and Biological Engineering  

DOI： 10.11239/jsmbe.51.M-32

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

Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na+-K+ cotransport and at high Na+ concentrations preferred Na+-selective transport, depending on the ionic conditions. But the physiological functions of this K+-transporting class II of HKT transporters remain unknown in plants, with the exception of the unique class II Na+ transporter OsHKT2;1. The genetically tractable rice (Oryza sativa; background Nipponbare) possesses two predicted K+-transporting class II HKT transporter genes, OsHKT2;3 and OsHKT2;4. In this study, we have characterized the ion selectivity of the class II rice HKT transporter OsHKT2;4 in yeast and Xenopus laevis oocytes. OsHKT2;4 rescued the growth defect of a K+ uptake-deficient yeast mutant. Green fluorescent protein-OsHKT2;4 is targeted to the plasma membrane in transgenic plant cells. OsHKT2;4-expressing oocytes exhibited strong K+ permeability. Interestingly, however, K+ influx in OsHKT2;4-expressing oocytes did not require stimulation by extracellular Na+, in contrast to other class II HKT transporters. Furthermore, OsHKT2;4-mediated currents exhibited permeabilities to both Mg2+ and Ca2+ in the absence of competing K+ ions. Comparative analyses of Ca2+ and Mg2+ permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKT1;1), Triticum aestivum HKT2;1 (TaHKT2;1), OsHKT2;1, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;1 mediate Mg2+ transport. Interestingly, cation competition analyses demonstrate that the selectivity of both of these class II HKT transporters for K+ is dominant over divalent cations, suggesting that Mg2+ and Ca2+ transport via OsHKT2;4 may be small and would depend on competing K+ concentrations in plants.

DOI： 10.1104/pp.110.168047

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.

DOI： 10.1093/pcp/pcr027

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

In order to confirm that mechanosensitive Ca2+ channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca2+ (Delta[Ca2+](c)). However, the observed Delta[Ca2+](c) decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (Delta E (m)) and stretching or compression of the plasma membrane. Significant Delta E (m) values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). Delta E (m) appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large Delta E (m) values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara.

DOI： 10.1007/s00232-009-9156-6

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Characean internodal cells generate receptor potential (DeltaE (m)) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated DeltaE (m) at the moment of both compression and decompression, and the amplitude of DeltaE (m) at the moment of decompression, (DeltaE (m))(E), was larger than that at compression. The long-lasting stimulus caused a membrane deformation (DeltaD (m)) having two components, a rapid one, (DeltaD (m))(rapid), at the moment of compression and a slower one, (DeltaD (m))(slow), during the long-lasting compression. We assumed that (DeltaD (m))(slow) might have some causal relation with the larger DeltaE (m) at (DeltaE (m))(E). We treated internodal cells with either HgCl(2) or ZnCl(2), water channel inhibitors, to decrease (DeltaD (m))(slow). Both inhibitors attenuated (DeltaD (m))(slow) during compression. Cells treated with HgCl(2) generated smaller (DeltaE (m))(E) compared to nontreated cells. On the other hand, cells treated with ZnCl(2) never attenuated (DeltaE (m))(E) but, rather, amplified it. Thus, the amplitude of (DeltaD (m))(slow) did not always show tight correlation with the amplitude of (DeltaE (m))(E). Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (DeltaE (m))(E). Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca(2+) channel.

DOI： 10.1007/s00232-007-9082-4

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Characean internodal cells generate receptor potential in response to mechanical stimuli. We studied responses of internodal cells to a long-lasting stimulus and the results were as follows. (i) The cell generated receptor potential at the moment of both compression and decompression. (ii) The receptor potential (DeltaE (m)) was significantly larger at the moment of decompression than at compression. (iii) The longer the duration of the stimulus, the larger was the magnitude of DeltaE (m) at the moment of decompression. (iv) Aequorin studies revealed that the increase in [Ca(2+)](c) (Delta[Ca(2+)](c)) took place at the moment of both compression and decompression. (v) The amplitude of Delta[Ca(2+)](c) was larger at the moment of decompression than at compression, as was the case for DeltaE (m). It was suggested that the amplitude of the receptor potential had a tight correlation with the degree of membrane deformation. We discussed the ionic mechanism of mechano-perception under a long-lasting stimulus in relation to mechanosensitive activation of Ca(2+) channels at the plasma membrane.

DOI： 10.1093/pcp/pci204

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

When an internodal cell of Chara corallina was stimulated with a mechanical pulse of various amplitudes lasting for 0.1 s (mechanical stimulus), the cell generated a receptor potential, which was highly dependent not only on the strength of the stimulus but also on the extracellular Cl- concentration. Extracellular Ca2+ was indispensable for generating receptor potential, since removal of Ca2+ reversibly inhibited generation of the receptor potential. The cytoplasmic Ca2+ level transiently rose upon mechanical stimulation. The stronger the mechanical stimulus, the larger was the increase in the cytoplasmic level of Ca2+. It is proposed that the first step of receptor potential is an activation of mechanosensitive Ca2+ channels at the plasma membrane.

DOI： 10.1093/pcp/pci004

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