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To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualise freezing behaviours in wintering Daphne kamtschatica var. jezoensis flower buds, which have naked florets and no bud scales. MRI images showed that anthers remained stably supercooled to the range from -14 to -21°C or lower while most other tissues froze by -7°C. Freezing of some anthers detected in MRI images between -14 and -21°C corresponded with numerous low temperature exotherms and also with the 'all-or-nothing' type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviours. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviours were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) while the remaining tissues tolerate extracellular freezing.

Summary We aimed to establish an in vitro differentiation procedure to generate matured small intestinal cells mimicking human small intestine from human-induced pluripotent stem cells (iPSCs). We previously reported the efficient generation of CDX2-expressing intestinal progenitor cells from embryonic stem cells (ESCs) using 6-bromoindirubin-3′-oxime (BIO) and (3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester (DAPT) to treat definitive endodermal cells. Here, we demonstrate the generation of enterocyte-like cells by culturing human iPSC-derived intestinal progenitor cells on a collagen vitrigel membrane (CVM) and treating cells with a simple maturation medium containing BIO, DMSO, dexamethasone, and activated vitamin D3. Functional tests further confirmed that these iPSC-derived enterocyte-like cells exhibit P-gp- and BCRP-mediated efflux and cytochrome P450 3A4 (CYP3A4)-mediated metabolism. We concluded that hiPS cell-derived enterocyte-like cells can be used as a model for the evaluation of drug transport and metabolism studies in the human small intestine., Highlights • hiPSC-derived intestinal progenitors are differentiated into enterocytes • The collagen vitrigel membrane supports hiPSCs to mature into functional enterocytes • iPSC-derived enterocytes show efflux transporter and metabolizing enzyme activities • iPSC-derived enterocytes can be used as a human small intestine model, We established a culture procedure to generate hiPSC-derived enterocyte-like cells that can be used as a model for the evaluation of drug transport and metabolism studies in the human small intestine.

Aim Organotypic brain slice culture preserves the geographical position of neurons and neuronal circuits. The slice cultures also maintain both non‐neuronal cell types and the surrounding extracellular matrix. The interface method has been widely used for slice cultures, in which brain slices are placed on semiporous polytetrafluoroethylene (PTFE) membranes. However, a low optical transparency of PTFE membrane makes it difficult to perform live imaging of deep regions of slice cultures using an inverted microscope. To overcome the issue, we evaluated the suitability of using collagen membranes for slice cultures, especially focusing on live imaging of the cellular dynamics of green fluorescent protein (GFP)‐expressing microglia. Methods Entorhinohippocampal slices were cultured on either collagen or PTFE membranes. The influence of membrane type on the ability to observe deep regions of slice cultures was examined by live imaging using an inverted microscope. Results Collagen membranes were thinner and had better optical transparency compared with PTFE membranes. There were no differences in cell viability, density of neurons or microglia. The densify of visible short branches of microglia in live imaging was higher in collagen membranes than PTFE membranes. Conclusion Collagen membranes are suitable for live imaging of cellular dynamics in slice cultures using an inverted microscope., Live imaging of organotypic slice cultures has been a useful method to study cell dynamics. One remaining issue with live imaging of slice cultures is the low transparency of commonly used polytetrafluoroethylene membranes. Here we report that slice culture membranes made of collagen can solve the issue of low transparency, facilitating live imaging of small cellular structures such as microglial processes.

To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualize freezing behaviors in wintering flower buds of Daphne kamtschatica var. jezoensis, which have no bud scales surrounding well-developed florets. MRI images showed that anthers remained stably supercooled to -14 ∼ -21°C or lower whilst most other tissues froze by -7°C. Freezing of some anthers detected in MRI images at ∼ -21°C corresponded with numerous low temperature exotherms and also with the “all-or-nothing” type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviors. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviors were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) whilst the remaining tissues tolerate extracellular freezing.

Laminin α chains (α1-α5 chains) are expressed in a tissue- and developmental stage-specific manner and have diverse chain-specific biological functions. Especially, laminin globular (LG) modules (LG1-LG5) located at the C-terminus of the α chains play a critical role in the biological activities of laminins. Each LG module is composed of a 14-stranded β-sheet (A-N) sandwich structure. We previously screened cell attachment activity of the loop regions between the E and F strands in the LG modules using 17 homologous peptides (EF peptides) and found that four active EF peptides bind to integrin α2β1. One of the four peptides, G4EF1 demonstrated improved cell attachment activity when cyclized. Here, we focused on the remaining three integrin α2β1-binding EF peptides (G5EF1, G3EF3, and G5EF5) and analyzed the relationship between their peptide conformation and cell attachment activity. First, we determined their active core sequences and found that G5EF1z (IGLEIVDGKVLFHVNN), G3EF3z (LLVTLEDGHIALST), and G5EF5z (KVLTEQVL) are the core sequences. Cyclic peptides of the core sequences (cycloG5EF1z, cycloG3EF3z, and cycloG5EF5z) enhanced integrin-mediated cell adhesion activity compared with their linear peptides. The results indicated that cell adhesion activity of the integrin α2β1-binding EF peptides is conformation dependent and that the loop structure is critical for their activity. This suggests that conformation of the loop regions plays an important role for the activities of the LG modules.

How plant tissues control their water behaviours (phase and movement) under subfreezing temperatures through adaptative strategies (freezing behaviours) is important for their survival. However, the fine details of freezing behaviours in complex organs and their regulation mechanisms are poorly understood, and non-invasive visualization/analysis is required. The localization/density of unfrozen water in wintering Cornus florida flower buds at subfreezing temperatures was visualized with high-resolution magnetic resonance imaging (MRI). This allowed tissue-specific freezing behaviours to be determined. MRI images revealed that individual anthers and ovules remained stably supercooled to -14 to -21 °C or lower. The signal from other floral tissues decreased during cooling to -7 °C, which likely indicates their extracellular freezing. Microscopic observation and differential thermal analyses revealed that the abrupt breakdown of supercooled individual ovules and anthers resulted in their all-or-nothing type of injuries. The distribution of ice nucleation activity in flower buds determined using a test tube-based assay corroborated which tissues primarily froze. MRI is a powerful tool for non-invasively visualizing unfrozen tissues. Freezing events and/or dehydration events can be located by digital comparison of MRI images acquired at different temperatures. Only anthers and ovules preferentially remaining unfrozen are a novel freezing behaviour in flower buds. Physicochemical and biological mechanisms/implications are discussed.

Control of freezing in plant tissues is a key issue in cold hardiness mechanisms. Yet freeze-regulation mechanisms remain mostly unexplored. Among them, ice nucleation activity (INA) is a primary factor involved in the initiation and regulation of freezing events in plant tissues, yet the details remain poorly understood. To address this, we developed a highly reproducible assay for determining plant tissue INA and noninvasive freeze visualization tools using MRI and infrared thermography. The results of visualization studies on plant freezing behaviors and INA survey of over 600 species tissues show that (1) freezing-sensitive plants tend to have low INA in their tissues (thus tend to transiently supercool), while wintering cold-hardy species have high INA in some specialized tissues; and (2) the high INA in cold-hardy tissues likely functions as a freezing sensor to initiate freezing at warm subzero temperatures at appropriate locations and timing, resulting in the induction of tissue-/species-specific freezing behaviors (e.g., extracellular freezing, extraorgan freezing) and the freezing order among tissues: from the primary freeze to the last tissue remaining unfrozen (likely INA level dependent). The spatiotemporal distributions of tissue INA, their characterization, and functional roles are detailed. INA assay principles, anti-nucleation activity (ANA), and freeze visualization tools are also described.

Control of freezing in plant tissues is a key issue in cold hardiness mechanisms. Yet freeze-regulation mechanisms remain mostly unexplored. Among them, ice nucleation activity (INA) is a primary factor involved in the initiation and regulation of freezing events in plant tissues, yet the details remain poorly understood. To address this, we developed a highly reproducible assay for determining plant tissue INA and noninvasive freeze visualization tools using MRI and infrared thermography. The results of visualization studies on plant freezing behaviors and INA survey of over 600 species tissues show that (1) freezing-sensitive plants tend to have low INA in their tissues (thus tend to transiently supercool), while wintering cold-hardy species have high INA in some specialized tissues; and (2) the high INA in cold-hardy tissues likely functions as a freezing sensor to initiate freezing at warm subzero temperatures at appropriate locations and timing, resulting in the induction of tissue-/species-specific freezing behaviors (e.g., extracellular freezing, extraorgan freezing) and the freezing order among tissues: from the primary freeze to the last tissue remaining unfrozen (likely INA level dependent). The spatiotemporal distributions of tissue INA, their characterization, and functional roles are detailed. INA assay principles, anti-nucleation activity (ANA), and freeze visualization tools are also described.

Ice nucleation activity (INA) of plant intrinsic origins is considered to play important roles in plant cold hardiness mechanisms. Yet, only a few studies have addressed the spatial and temporal localization of plant INA, how it is regulated and what its functional roles are. In our previous study ( Kishimoto et al., 2014 ), we revised a test tube method and developed a highly reproducible assay for measuring INA of plant specimens and demonstrated that high INA occurred in the cell wall fraction of wintering bark tissues of blueberry stems and corresponded well to the freezing behavior (extracellular freezing) of the stem bark. Here, we followed precisely seasonal changes in the stem INA of two blueberry cultivars and alterations in the stem INA caused by artificial incubation at various low temperatures. INA of newly developed shoots was low but increased rapidly by July when the stem became seemingly matured, then gradually increased with the maximum in October or early November just before the first autumnal frost. Following the subsequent recurrent frosts, the stem INA gradually decreased. This tendency was consistent between the two cultivars differing in the level of cold hardiness. INA in the stems of September until February was increased by incubation at 0-7 °C whilst decreased by freezing to lower temperatures. The in vitro results corroborate the seasonal changes in the stem INA in the field but the mechanisms remain to be investigated. The highest level of INA (expressed as the median ice nucleation temperature) observed with current year stems (7.5 mm-long) of Woodard in October of 2010-2013 was -0.9 ∼ -1.0 °C when determined with 2 mL assay system (-1.1 ∼ -1.3 °C with 0.5 mL system). This may likely be one of the highest INA of biological origins ever reported.

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.

Hollow particles have attracted considerable attention owing to their unique properties. In this work, hollow zirconia particles were synthesized using rod-shaped gram-negative bacteria, Escherichia coli , as templates. A zirconia precursor, generated by the hydrolysis of zirconium butoxide, was deposited on the surface of the bacterial cells to form the shell of the hollow particles. The as-synthesized particles had the morphology of the bacterial templates, and were about 1.7 μm long and 0.8 μm across. The bacterial templates could be removed by calcination at 800 °C. The particles shrank on calcination to a final size of about 1.0 μm long and 0.4 μm across, with a wall thickness of about 69 nm. The specific surface area and average pore diameter were 45.7 m 2 /g and 1.9 nm, respectively. When fixed cells without internal water were used as templates, no hollow particles were observed; this implies that the internal water inside the cells acted as the initiator for the hydrolysis of zirconium butoxide.

How plant tissues control their water behaviours (phase and movement) under subfreezing temperatures through adaptative strategies (freezing behaviours) is important for their survival. However, the fine details of freezing behaviours in complex organs and their regulation mechanisms are poorly understood, and non-invasive visualization/analysis is required. The localization/density of unfrozen water in wintering Cornus florida flower buds at subfreezing temperatures was visualized with high-resolution magnetic resonance imaging (MRI). This allowed tissue-specific freezing behaviours to be determined. MRI images revealed that individual anthers and ovules remained stably supercooled to -14 to -21 °C or lower. The signal from other floral tissues decreased during cooling to -7 °C, which likely indicates their extracellular freezing. Microscopic observation and differential thermal analyses revealed that the abrupt breakdown of supercooled individual ovules and anthers resulted in their all-or-nothing type of injuries. The distribution of ice nucleation activity in flower buds determined using a test tube-based assay corroborated which tissues primarily froze. MRI is a powerful tool for non-invasively visualizing unfrozen tissues. Freezing events and/or dehydration events can be located by digital comparison of MRI images acquired at different temperatures. Only anthers and ovules preferentially remaining unfrozen are a novel freezing behaviour in flower buds. Physicochemical and biological mechanisms/implications are discussed.

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.

Laminins, a multifunctional protein family of extracellular matrix, interact with various types of integrin. Here, integrin-mediated cell adhesive peptides have been systematically screened in the laminin α4 and α5 chain G domain peptide library consisting of 211 peptides by both the peptide-coated plastic plates and peptide-conjugated Sepharose bead assays using human dermal fibroblasts. Thirteen peptides promoted cell spreading and the activity was specifically inhibited by EDTA. Cell attachment to 11 peptides was inhibited by anti-integrin β1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin α3 and α6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin α3β1- and α6β1-mediated cell attachment. Further, most of the integrin-mediated cell adhesive peptides are located in the loop regions in the G domains, suggesting that structure is important for the integrin specific recognition. Integrin binding peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development.

In this paper, we test whether corporate managers have the ability to time the market, which is still controversial in the corporate finance literature. In empirical analyses, we show that firms conduct share repurchase programs when stock prices decreased in the previous month and that firms conducting market share repurchase programs outperform the market over the subsequent months. The evidence implies that corporate managers are informed and/or skillful at detecting mispricing in the stock markets.

This study examines the relationship between managerial overconfidence and corporate financing decisions by constructing proxies for managerial overconfidence based on the track records of earnings forecasts in Japanese listed firms. We find that managers have the stable tendency to forecast overly upward earnings compared to actual ones and that their upward bias decreases the probability of issuing equity in the public market by about 4.7 percent per one standard error, which economically has the strongest impact on financing decisions. This tendency is observed when we employ alternative measures for managerial overconfidence and other model specifications. However, in private placements, the choice to offer equity is not always avoided by managers. This implies that managers place private equity with the expectation of the certification effect

Hollow inorganic particles have attracted great interest because of their unique physicochemical properties. In this study, hollow silica microparticles were prepared using a rod-shaped gram-negative bacterium, Escherichia coli (KP7600), as a biological template. Silica nanoparticles were generated in addition to coated biological templates when the reaction rate was increased, so control of reaction rate is important for coating silica smoothly onto the bacterial surface. Silica coating was also carried out using the fixed cells (with and without internal water) using glutaraldehyde as templates. When the fixed cells without internal water were used as templates, no rod-shaped particles were observed after calcination of the synthesized particles. By contrast, silica hollow particles were formed using the fixed cells with internal water as templates. This means that the internal water inside biological cells acts as an initiator for hydrolysis of tetraethyl orthosilicate (TEOS) and results in the formation of smooth silica shell surface and indicates that the use of dry cultured bacteria templates is not required. Thus, there is a significant benefit in using gram-negative bacteria as templates for producing hollow silica microparticles, compared with the method using dried gram-positive bacteria templates.

Background: It is unclear how much the sympathetic nervous system is involved in the development of pulmonary arterial hypertension (PAH). The present study examined whether or not a pure α/β-adrenergic receptor blocker (arotinolol) could prevent the development of PAH and right ventricular hypertrophy (RVH) in a rat model of monocrotaline (MCT)-induced PAH. Methods and Results: The heart rate, arterial blood pressure (BP), left ventricular pressure, pulmonary artery pressure (PAP), and right ventricular pressure (RVP) were measured after administration of arotinolol or saline for 2 weeks. Ventricular weight and myocyte size were also measured. Mean PAP was increased less in the arotinolol group (n=6), (53 ±9 vs 21 ±2 mmHg in the control (n=6); P

Constant relative humidity (RH) chambers using phosphoric acid (PA) are useful for controlled desiccation of pollen and in vitro cultured materials for cool-dry storage or cryopreservation (Ishikawa et al., 2005, Ishikawa and Oda, 2008). Here we demonstrate that the chambers are also convenient for accelerated ageing of seeds. Rice seeds 3-11 years old (stored at 8-9°C) were artificially aged for 0-41 days in PA-assisted relative humidity (RH) chambers (RH 93%) placed in an incubator set at 37°C. At designated time periods during ageing, vigour and longevity of the seeds with different storage history were evaluated by monitoring germination timecourse and seedling shoot and root growth. Under these ageing conditions, seeds harvested in 2002 had half germinability (50% longevity) period of 33 days while older seeds (stored for longer periods) showed faster decline in germinability, longer lag time before germination, slower rates of germination and reduced shoot and root growth. Root growth was more sensitively affected by storage and accelerated ageing than shoot growth. PA-assisted system is simple, economical and safer than conventional RH chamber systems using saturated solutions or H2SO4 (Winston and Bates, 1960, Solomon, 1951). Various constant RH are available at desired temperatures using a single compound and convenient for realizing controlled deterioration of seeds at various RH and temperature regimes.

Relative humidity (RH) in constant RH chambers using various concentration of phosphoric acid (PA) was re-determined with a recently developed hygrometer (HygroLog NT2-D equipped with a sensor HygroClip S, ROTRONIC AG), which is superior in accuracy. Newly determined RH-PA concentration profiles were reverse semi-sigmoidal and very similar at 25 and 10°C. Differences in RH values between the present and previous determinations were larger at PA concentrations of 0-50% and 90-100%, primarily due to the property of the hygrometers employed. Accordingly, amendments were made to all the RH values in the previous paper (Ishikawa et al., 2005). This will make the PA-based constant RH system more accurate and reliable and ready to be used for various purposes.

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl2, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds.

Water-saving rice-winter crop rotation systems were repeated for 4 cycles from 2000 to 2004 in an urban area, Nishitokyo, Japan, to assess the effects of water-saving (i.e. non-flooded vs. flooded) on grain yield of rice (Oryza sativa L.) and chemical constituents of percolating water. The effects of pre-rice winter cropping compared with fallow on rice yield were also examined. The pre-cultivated crops were wheat (Triticum aestivum L.), italian ryegrass (Lolium multiflorum Lam.) or spinach (Spinacea oleracea L.) with their above-ground parts removed, chinese milk vetch (Astragalus sinicus L.) or rapeseed (Brassica napus L.) with their above-ground parts incorporated before rice transplanting. Neither winter cropping effects nor its interaction with water-saving were significant for rice yield, although the yield after rapeseed incorporation tended to be 9 % higher than that after fallow. In 2001, 2003 and 2004, when more than 70% of irrigation water was saved in the non-flooded trial, average yield in non-flooded trial was 58 % of flooded trial, but water productivity increased (from 0.10 to 0.16 kg m-3). Among the 3 years, yield in non-flooded trial was highest in 2004 when the amounts of irrigation and total water supply was larger, the frequency of dry spells was the lowest, and 2 seedlings were transplanted per hill. The nitrate and nitrite concentrations in the percolating water were far below the environmental standard values by WHO. The study showed that incorporation of winter crops had no negative effects on water-saving rice production at least for the first 4 years, and that under extreme water-saving, irrigation and planting methods could minimize yield reduction.

Wintering flower buds of cold hardy Rhododendron japonicum cooled slowly to subfreezing temperatures are known to undergo extraorgan freezing, whose mechanisms remain obscure. We revisited this material to demonstrate why bud scales freeze first in spite of their lower water content, why florets remain deeply supercooled and how seasonal adaptive responses occur in regard to extraorgan freezing in flower buds. We determined ice nucleation activity (INA) of various flower bud tissues of using a test tube-based assay. Irrespective of collection sites, outer and inner bud scales that function as ice sinks in extraorgan freezing had high INA levels whilst florets that remain supercooled and act as a water source lacked INA. The INA level of bud scales was not high in late August when flower bud formation was ending, but increased to reach the highest level in late October just before the first autumnal freeze. The results support the following hypothesis: the high INA in bud scales functions as the subfreezing sensor, ensuring the primary freezing in bud scales at warmer subzero temperatures, which likely allows the migration of floret water to the bud scales and accumulation of icicles within the bud scales. The low INA in the florets helps them remain unfrozen by deep supercooling. The INA in the bud scales was resistant to grinding and autoclaving at 121°C for 15 min, implying the intrinsic nature of the INA rather than of microbial origin, whilst the INA in stem bark was autoclaving labile. Anti-nucleation activity (ANA) was implicated in the leachate of autoclaved bud scales, which suppresses the INA at millimolar levels of concentration and likely differs from the colligative effects of the solutes. The tissue INA levels likely contribute to the establishment of freezing behaviors by ensuring the order of freezing in the tissues: from the primary freeze to the last tissue remaining unfrozen.

� Background and Aims Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing. � Methods In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0� 1 M sucrose at 25 � C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0� 4 M and 0� 7 M sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied. � Key Results During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0� 1 M sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2� 4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels. � Conclusions These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.

Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS) and green fluorescent protein (GFP) genes were co-cultivated for 3 days with bromegrass cells at the late exponential or early stationary growth phase (7–9 days after subculture). These conditions gave optimal transformation efficiency. Putative transformants were identified by selection for geneticin resistance and by examining the calluses using fluorescence microscopy. This allows the elimination of escapes and selection of cells that express the target genes. PCR and Southern blot analyses confirmed the integration of the GUS and GFP genes into the genome of transformed bromegrass cell lines. Transformants with various levels of GUS expression were obtained with a high frequency following Agrobacterium-mediated gene transfer and visual selection by GFP. The successful transformation method described allows reverse genetics approaches for analyzing cold hardiness genes isolated from bromegrass cells.

For successful storage and cryopreservation of recalcitrant (short-lived or desiccation-sensitive) plant seeds, excised seed embryos or pollen, precise control of water content and/or slow drying rates are often crucial. To realize such controlled desiccation, we developed simple and safe constant relative humidity (RH) chambers using a single compound, phosphoric acid (PA). It consisted of a bowl of PA-water solution placed in a hermetically sealed box. With this apparatus, RH ranges of 4-88% were obtained from 100-10% PA solutions, respectively. Similar RH-PA concentration profiles were obtained at 24 and 7°C. Constant RH was ensured at a constant temperature as temperature fluctuations resulted in mirror-image RH oscillations within the chamber. When the temperature was greatly changed within 5-30°C ranges, the chamber regained the same equilibrium RH in 4-8 h. Using this system, typical recalcitrant winter rye pollen was successfully stored under RH50% at 7°C for 10 days. Alginate beads, irrespective of sucrose concentrations (0.7-1.2 M), were desiccated to equilibrium water contents of 11.5-32% (fresh weight basis) under constant RH of 27-78% at 24°C. This system is simple, economical and safer than conventional RH chamber systems using saturated solutions or sulfuric acid. It allows constant RH available at various temperatures and is useful for controlled desiccation of small recalcitrant genetic or in vitro resources for dry-cold or cryogenic storage.

Alginate beads containing axillary buds of in vitro-grown gentian (Gentiana scabra Bunge var. buergeri Maxim.), were successfully cryopreserved following 2 step-preculture with sucrose and desiccation. The optimal preculture conditions were as follows: axillary buds were excised from in vitro-grown gentian plants and precultured on semi-solid Murashige and Skoog (MS) medium containing 0.1 M sucrose for 10 days (25 °C, 16-h photoperiod) (first step). This was followed by incubation on semi-solid MS media containing 0.4 M (1 day) and then 0.7 M sucrose (1 day) (second step). After preculture, the buds were encapsulated in alginate beads and desiccated aseptically on silica gel for 9 h to a water content of 10% (fresh weight basis), followed by immersion in liquid nitrogen (LN). With this protocol, 87% of the gentian buds survived exposure to LN and showed normal development of shoots and roots in vitro and in vivo. Depletion of NH4NO3 in the regeneration medium did not improve survival following desiccation and exposure to LN. The results show that 2 step-preculture with sucrose is effectively applicable in encapsulation–desiccation based cryopreservation of gentian axillary buds. This preculture can replace the conventionally used lengthy cold-hardening treatment and is useful for routine cryopreservation of gentian germplasm.

We noted that ammonia nitrogen was not adsorbed by the cultivated layers of highly permeable paddy fields during the initial fertilization period, but reached the lower layers relatively early. In our study, we considered an exponential equation from an aqua-environmental perspective with the goal of obtaining good growth of rice plants in order to estimate the concentrations and integrated volume of ammonia nitrogen accompanying paddy percolation. Using this exponential equation, we were able to derive a relation between time and concentrations of paddy percolation water, and hypothesized that if percolation rates were less than 10 mm/day, percolation would have no effect on rice growth, while simultaneously helping to maintain the good water quality of the extra-paddy environment. We also clarified the differences between the potential ammonia nitrogen adsorption volume derived from the CEC value and the integrated amount of ammonia nitrogen water in soil, and considered the causes from the perspectives of solute movement and water movement.

The association between disorders of thyroid function and psychiatric symptoms is well established. Generally, hypo-thyroidism is recognized as being able to induce psychotic symptoms. However, psychotic symptoms without affective and cognitive disturbances are rare in hyperthyroidism. We presented a patient with Graves’ disease who suffered from delusions and hallucinations without affective and cognitive disturbances following abrupt normalization of thyroid function. Furthermore, the patient was clinically and biochemically euthyroid when the psychotic symptoms developed. There was resolution of psychotic symptoms in this case. It is suggested that the abrupt change of thyroxine may cause susceptible individuals to become transient psychotic state.

One may have seen wintering rosette leaves totally frozen and wilted in the early morning but recover during the daytime. How can cold hardy plants survive freezing of the tissues, unlike animal tissues? Cold hardy plants seem to have evolved various strategies. One example is extracellular freezing, where icicles primarily form in intercellular spaces whilst the cells are dehydrated, yet the underlying mechanisms remain unclear. In this study, using blueberry stems, we found high ice nucleation activity specifically localized in the cell wall fraction of bark tissues. This activity likely contributes to the primary and spontaneous initiation of freezing in the intercellular spaces of the bark to successfully perform extracellular freezing., Controlled ice nucleation is an important mechanism in cold-hardy plant tissues for avoiding excessive supercooling of the protoplasm, for inducing extracellular freezing and/or for accommodating ice crystals in specific tissues. To understand its nature, it is necessary to characterize the ice nucleation activity (INA), defined as the ability of a tissue to induce heterogeneous ice nucleation. Few studies have addressed the precise localization of INA in wintering plant tissues in respect of its function. For this purpose, we recently revised a test tube INA assay and examined INA in various tissues of over 600 species. Extremely high levels of INA (−1 to −4 °C) in two wintering blueberry cultivars of contrasting freezing tolerance were found. Their INA was much greater than in other cold-hardy species and was found to be evenly distributed along the stems of the current year's growth. Concentrations of active ice nuclei in the stem were estimated from quantitative analyses. Stem INA was localized mainly in the bark while the xylem and pith had much lower INA. Bark INA was located mostly in the cell wall fraction (cell walls and intercellular structural components). Intracellular fractions had much less INA. Some cultivar differences were identified. The results corresponded closely with the intrinsic freezing behaviour (extracellular freezing) of the bark, icicle accumulation in the bark and initial ice nucleation in the stem under dry surface conditions. Stem INA was resistant to various antimicrobial treatments. These properties and specific localization imply that high INA in blueberry stems is of intrinsic origin and contributes to the spontaneous initiation of freezing in extracellular spaces of the bark by acting as a subfreezing temperature sensor.

This paper concerns the surficial integration of the farmland by right to use setting in the land consolidation. The success example of the surficial integration of right to use was investigated, and the success factor was investigated from ground, drawing of the replotting and hearing investigation. The reason why the surficial integration of right to use succeeded in this region is unified in the consciousness that the whole colony should change the agricultural business of large scale, and it is because the organization which adjusts right to use setting of colony agricultural joint council functioned.

The purpose of this study is the better suggetion for the farm inn management. The farm inn management hindrance was grasped by the interview investigation to the manager, and the requirement for the user was grasped by the questionnairing to the lodger in the form of the satisfaction. Unimprovement of relades law and regulation, transportation expenses, unimprovement of information source were raised as a hindrance. The value in which landscape and building and facilities were high on the satisfaction was shown. And result of the multiple regression analysis, at the satisfaction of the whole travel, it was confirmed that the satisfaction of the landscape gave the effect most.

A simple and efficient procedure for the transient permeabilization of cultured cells has been developed for the incorporation of a high molecular weight substance, fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (FITC-IgG, 150 kDa) into Nicotiana tabacum BY-2 cells grown in suspension cultures. The cells were treated with cell permeabilization solutions (CPS) comprising varying concentrations of glycerol (Gly), sucrose (Suc), ethylene glycol (EG) and dimethyl sulfoxide (DMSO). When the cells were permeabilized by treating with CPS-3 (Gly:Suc:EG:DMSO=20:5:20:5, w/v%) containing FITC-IgG for 15 s at room temperature followed by washing with ice-cold 1 M sucrose and modified LS medium, 76% of the cells incorporated FITC-IgG and 78% of the cells remained viable according to fluorescein diacetate (FDA) staining. Most treated cells retained intact internal structure and the incorporated FITC-IgG was detected in cytoplasm and in the nuclear region. Cells of different ages were studied. Four day-old cells were found suitable for the incorporation of FITC-IgG and cell survival. Inclusion of 10 mM CaCl 2 in CPS-3 improved both FITC-IgG uptake and cell survival. The regrowth of treated cultures lagged for the first 3 days, then followed a growth pattern similar to the untreated controls.

Axillary buds of in vitro-grown gentian ( Gentiana scabra Bunge var. buergeri Maxim.) were successfully desiccated and cryopreserved using a novel two-step preculture method: preculturing axillary buds excised from in vitro-grown gentian plants on Murashige and Skoog (MS) semi-solid medium containing 0.1 M sucrose for 8–13 days at 25°C under a 16 h-photoperiod (first step) and the subsequent short period incubations on 0.4 M sucrose (MS agar medium) for 1 day followed by 0.7 M sucrose (MS agar medium) for 1 day (second step). After preculturing, the buds were air-dried down to about 10% water content (fresh weight basis) without encapsulation, and then immersed in liquid nitrogen (LN). With this cryogenic protocol, we could obtain 78–90% of survival of axillary buds following desiccation and LN exposure. Most of the buds that survived showed normal shoot and root development. The most important part of this protocol was the preculture which circumvents the conventionally used lengthy cold-hardening treatment. The results show that induction of desiccation tolerance by preculturing was specific to a few species of sugars used in the medium and not merely caused by osmotic effects. The efficient desiccation–cryopreservation protocol shown here using the new preculture method appears promising for routinely cryopreserving gentian germplasm.

1H-Nuclear magnetic resonance (NMR) microscopy was used to study the freezing behavior of wintering buds of full-moon maple (Acer japonicum Thunb.). The images obtained predominantly reflected the density of mobile (i.e. non-ice) protons from unfrozen water. A comparison of NMR images taken at different subfreezing temperatures revealed which tissues produced high- and low-temperature exotherms in differential thermal analyses. In leaf and lower buds of A. japonicum, the scales and stem bark tissues were already frozen by -7[deg]C, but the primordial inflorescence and terminal primordial shoots remained supercooled at -14[deg]C, and the lateral primordial shoots were unfrozen even at -21[deg]C. The freezing of these supercooled tissues was associated with their loss of viability. The size of the supercooled primordial shoots and inflorescences was gradually reduced with decreasing temperature, indicating extraorgan freezing in these tissues. During this process the formation of dark regions beneath the primordia and subsequent gradual darkening in the basal part of supercooled primordia were visible. As the lateral shoot primordia were cooled, the unfrozen area was considerably reduced. Since the lateral primordia remained viable down to -40[deg]C, with no detectable low-temperature exotherms, they probably underwent type I extraorgan freezing. Deep supercooling in the xylem was clearly imaged. NMR microscopy is a powerful tool for noninvasively visualizing harmonized freezing behaviors in complex plant organs.

Tissue-cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) were successfully cryopreserved in liquid nitrogen (LN) using a slow prefreezing method. The highest survival and recovery were obtained with the following procedure. Three week-old shoot primordia clumps were dissected into pieces of 2-3 mm of diameter and precultured in standard medium for 3 days. They were directly soaked in CSP1 cryoprotective solution (10%w/v sucrose, 10%w/v dimethylsulfoxide and 5%w/v glycerol) and incubated at room temperature for 30 min. Samples were ice-inoculated at -8 °C and cooled at a rate of between 0.3 and 1 °C min−1 with a programmable freezer to -30 °C for prefreezing. They were then plunged into LN for storage. After rapid thawing in 40 °C water, the cryoprotective solution was slowly diluted 5 fold in a dropwise manner with 3% sucrose and the shoot primordia were transferred onto regeneration medium. Under optimal conditions, more than 80% of cryopreserved shoot primordia were viable and 50 to 80% regenerated shoots after one month of reculture. Cryopreserved shoot primordia could be used both for reproducing a shoot primordia culture and for regenerating plants.

Background The role of abscisic acid (ABA) as a possible activator of cold acclimation process was postulated since endogenous levels of ABA increase temporarily or constitutively during cold-hardening. Exogenous application of ABA has been known to induce freezing tolerance at ambient temperatures in in vitro systems derived from cold hardy plants. Yet, some cell cultures acquired much greater freezing tolerance by ABA than by cold whilst maintaining active growth. This raises questions about the relationships among ABA, cold acclimation and growth cessation. To address this question, we attempted to 1) determine whether exogenous ABA can confer freezing tolerance in chilling-sensitive rice suspension cells and seedlings, which obviously lack the mechanisms to acquire freezing tolerance in response to cold; 2) characterize this phenomenon by optimizing the conditions and compare with the case of cold hardy bromegrass cells. Results Non-embryogenic suspension cells of rice suffered serious chilling injury when exposed to 4°C. When incubated with ABA at the optimal conditions (0.5-1 g cell inoculum, 75 μM ABA, 25-30°C, 7–10 days), they survived slow freezing (2°C/h) to −9.0 ~ −9.3°C (LT50: 50% killing temperature) while control cells were mostly injured at −3°C (LT50: -0.5 ~ −1.5°C). Ice-inoculation of the cell suspension at −3°C and survival determination by regrowth confirmed that ABA-treated rice cells survived extracellular freezing at −9°C. ABA-induced freezing tolerance did not require any exposure to cold and was best achieved at 25-30°C where the rice cells maintained high growth even in the presence of ABA. ABA treatment also increased tolerance to heat (43°C) as determined by regrowth. ABA-treated cells tended to have more augmented cytoplasm and/or reduced vacuole sizes compared to control cultures with a concomitant increase in osmolarity and a decrease in water content. ABA-treated (2–7 days) in vitro grown seedlings and their leaves survived slow freezing to −3°C with only marginal injury (LT50: -4°C) whereas untreated seedlings were killed at −3°C (LT50: -2°C). Conclusions The results indicate that exogenous ABA can induce some levels of freezing tolerance in chilling-sensitive rice cells and seedlings, probably by eliciting mechanisms different from low temperature-induced cold acclimation.