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Percutaneous coronary intervention (PCI) requires multiple staff members, including interventional cardiologists, with the physical burden of heavy protective measures to minimize radiation exposure. Here, we aimed to investigate the safety of task sharing with clinical engineers (CEs) working as 1st assistant during ad hoc PCI. We retrospectively included 286 patients who underwent ad hoc PCI following diagnostic catheterization for coronary artery disease between April 2019 and March 2021. Procedural complications including coronary perforation or rupture, myocardial infarction, cerebral embolism, cardiovascular death, decreased kidney function, and radiation parameters were compared between the two clinical settings [CE group, CEs as the 1st assistant from the beginning of diagnostic coronary angiography to the end of PCI vs. doctor (DR) group, others]. There was no increase in the ratio of procedural complications in the CE group (1.7%) versus the DR group (1.2%). Fluorescence time and radiation exposure dose were significantly reduced in the CE group {25 min [interquartile range (IQR), 19–35 min] vs. 28 min (IQR, 20–39 min), P = 0.036; 908 mGy (IQR, 654–1326 mGy) vs. 1062 mGy (IQR, 732–1594 mGy), P = 0.049}. The median amount of contrast medium was significantly reduced in the CE group [100 mL (IQR, 80–119 mL) vs. 110 mL (IQR 90–140 mL), P

Adipose-derived regenerative cell (ADRC) is a promising alternative source of autologous somatic stem cells for the repair of damaged tissue. This study aimed to assess the safety and feasibility of autologous ADRC implantation for therapeutic angiogenesis in patients with critical limb ischaemia (CLI). A clinical pilot study—Therapeutic Angiogenesis by Cell Transplantation using ADRCs (TACT-ADRC) study—was initiated in Japan. Adipose tissue was obtained by ordinary liposuction method. Isolated ADRCs were injected into the ischaemic limb. We performed TACT-ADRC procedure in five patients with CLI. At 6 months, no adverse events related to the TACT-ADRC were observed. No patients required major limb amputation, and ischaemic ulcers were partly or completely healed during the 6-month follow-up. In all cases, significant clinical improvements were seen in terms of rest pain and 6-min walking distance. Numbers of circulating CD34+ and CD133+ cells markers of progenitor cell persistently increased after ADRC implantation. The ratio of VEGF-A165b (an anti-angiogenic isoform of VEGF) to total VEGF-A in plasma significantly decreased after ADRC implantation. In vitro experiments, cultured with ADRC-conditioned media (CM) resulted in increased total VEGF-A and decreased VEGF-A165b in C2C12 cells, but not in macrophages. ADRC-CM also increased CD206+ cells expression and decreased TNF-α in macrophages. Autologous ADRC implantation was safe and effective in patients with CLI and could repair damaged tissue via its ability to promote angiogenesis and suppress tissue inflammation.

We aimed to investigate the impact of post-discharge scheduled hospital visits on readmission due to heart failure (HF). In this retrospective study, a total of 245 patients (N = 101 in the scheduled hospital visit group, N = 144 in the non-scheduled hospital visit group) who were alive with free from readmission due to HF for 90 days after discharge were enrolled. The patients had been hospitalized with acute decompensated HF between August 2018 and July 2019. Scheduled hospital visits were recommended 90 days after the patients had been discharged. After checking their self-care adherence, nurse-led self-care maintenance and monitoring were provided. To determine the effectiveness of the scheduled hospital visits, we conducted landmark analyses divided into two periods: Scheduled visits within 180 days, and after 180 days. The readmission rate due to HF within 180 days was lower in the scheduled visit group. In the landmark analysis, the 1-year incidence rate of readmission was significantly lower in patients with a scheduled hospital visit than in those without, in the period within 180 days (2.0% vs 9.0%

Therapeutic angiogenesis with autologous stem/progenitor cells is a promising novel strategy for treatment of severe ischemic diseases. Human clinical trials utilizing autologous adipose-derived regenerative cells (ADRCs) have not reported treatment-related critical adverse effects thus far. However, there is still a large knowledge gap regarding whether treatment of ischemic diseases with angiogenic therapy using ADRCs would promote unfavorable angiogenesis associated with tumors in vivo. Herein, we addressed this clinical question using a mouse hindlimb ischemia (HLI) and simultaneous remote tumor implantation model. C57BL/6J background wild-type mice were injected with murine B16F10 melanoma cells on their back, 1 day before ischemic surgery. These mice were subjected to surgical unilateral hindlimb ischemia, followed by ADRC implantation or PBS injection into the hindlimb ischemic muscles on the next day. Intramuscular implantation of ADRCs enhanced tissue capillary density and blood flow examined by a laser Doppler blood perfusion analysis in hind limb. However, this therapeutic regimen for ischemic limb using ADRCs did not affect remote melanoma growth nor the density of its feeder artery, angiogenesis, and lymphatic vessels compared with the PBS group. In addition, no distant metastases were detected in any of the mice regardless of the group. In conclusion, local implantation of ADRCs promotes angiogenesis in response to tissue ischemia in the hindlimb without promoting remote tumor growth and related angio/lymphangiogenesis. Therapeutic angiogenesis to the ischemic hindlimb using ADRCs seems to be safe regarding remote tumor growth.

This paper presents a virtual reality (VR) system of evacuation in the cases of various disasters. We considered a combination of disasters under realistic scenarios. Disaster simulations by numerical models were imported into the VR system to express a realistic situation. Not only disaster experts but also designers and non-professionals are able to share realistic experiences for escape from the disasters. This system is useful for performance-based design, planning of escape, disaster prevention, evacuation drill etc. The VR system consists of Building Information Modelling, physical simulation models for disasters and human behaviour’s simulation.

Background Although physical restraint is still used in psychiatric inpatient settings, it sometimes causes serious side effects, including deep vein thrombosis (DVT) and resulting pulmonary embolism. Objective The aim of this study was to investigate the incidence of the DVT in restrained patients who were receiving routine prophylaxis and to identify the risk factors of this condition. Methods This study was conducted at Sakuragaoka Memorial Hospital, Japan from December 2008 to September 2010. Inpatients who were restrained during the study period were included. All restrained patients wore graduated compression stockings and were recommended to receive subcutaneous injection of unfractionated heparin during the period of restraint unless it was contraindicated. When plasma d-dimer level at the time of removal of restraint was ≥ 0.50μg/dL, the patients underwent a Doppler ultrasound scanning of their lower extremities to examine the presence of DVT. A multiple logistic regression model was used to examine the effects of demographic and clinical characteristics on the incidence of DVT. Results A total of 181 patients (98 men; mean ± standard deviation age, 47.8 ± 17.0y) were included; DVT was detected in 21 patients (11.6%). A longer duration of restraint (odds ratio=9.77, 95% confidence interval=1.56–61.03, p = 0.015), excessive sedation (odds ratio=4.90, 95% confidence interval=1.33–18.02, p = 0.017), lower antipsychotic dosage (odds ratio=0.05, 95% confidence interval=0.005–0.57, p = 0.016), and recent medical hospitalization (odds ratio=11.44, 95% confidence interval=2.13–61.47, p = 0.004) were significantly associated with the incidence of DVT. Conclusion The incidence of DVT in restrained psychiatric patients was not low in spite of prophylaxis. These findings emphasize the importance of regular screening of and thorough assessments of DVT, especially in restrained psychiatric patients.

This study aimed to clarify the effect of interactions between membranes and natural organic matter (NOM), which is considered to be the major membrane foulant in wastewater treatment processes. Sodium alginate (SA), humic acid (HA) and bovine serum albumin (BSA) were used as NOM models. Hollow fiber membranes were prepared using cellulose acetate butyrate polymer (CAB). Filtration experiments were carried out using SA, HA and BSA solutions and the membrane fouling behavior was examined. NOM adsorption on CAB membranes was measured by quartz crystal microbalance with dissipation (QCM-D), where NOM solutions were flowed across CAB spin-coated quartz crystal sensors and adsorbed. SA showed more severe permeate flux decline during the early stage of filtration and lower recovery of permeate flux after backwashing compared with HA. The severe flux decline for SA was due to pore-plugging and cake formation with high molecular weight components. The BSA solution, with no high molecular components, showed a gradual permeate flux decline and resulted in lower permeate flux after 240 min filtration compared with HA. The gradual permeate flux decline with the BSA solution was due to ready adsorption of BSA on the CAB membrane.

The object of this study is to clarify the effect of membrane surface morphology on membrane fouling with sodium alginate (SA) which is a kind of natural organic matter (NOM) and is considered to be the main foulant in membrane water treatment. Cellulose acetate butyrate polymer which is superior in heat-resistance and mechanical strength was used as membrane material. Four kinds of membranes having different surface and cross-sectional structures were prepared via thermally induced phase separation and non-solvent induced phase separation. In the case of the filtration of 50 mg L −1 SA solution from the outer surface to inner surface, the membrane having the dense outer surface showed higher permeability and higher recovery of permeability by backwashing. An addition of calcium chloride into the sodium alginate solution resulted in significant flux decline due to thick cake layer formed by cross-linking of sodium alginate. In this case, the membranes with the dense outer surface also showed higher permeability. Furthermore, it was found that membrane fouling with sodium alginate took place mainly on the outer surface rather than inside the membrane by measuring infrared absorption spectrum of the membrane after filtration of sodium alginate solution with and without calcium chloride.

In this work, the relationship between surface roughness of the membrane and membrane fouling was investigated with sodium alginate which is a kind of extracellular polymeric substances (EPS) and is regarded as a main foulant in membrane bioreactor (MBR). Cellulose acetate butyrate polymer which is superior in heat-resistance and mechanical strength was used as membrane material. The membranes having the gear-shaped structures of different heights on the outer surface were prepared by using the several spinnerets via thermally induced phase separation. The membrane having the higher projection on the outer surface showed higher antifouling property. The recovery of the permeability by backwashing also increased by the higher projection of the outer surface of the membrane. Although the membrane permeability decreased by adding calcium chloride into the alginate solution, the membrane with the higher projection also showed higher permeability and recovery by backwashing. The increase of the permeability and the recovery by backwashing was not owing to the increase of the outer surface area by the gear-shaped structure formation. From the observation of the membrane outer surface after filtration of sodium alginate and humic acid, it was revealed that foulants were accumulated in the valley between projections, while the top of the projection was clean. This is the reason for the higher antifouling property for the membrane with the higher projection structure.

ABA is an important phytohormone regulating various plant processes, including stress tolerance, seed development and germination. SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 are redundant ABA-activated SNF1-related protein kinases 2 (SnRK2s) in Arabidopsis thaliana. We examined the role of these protein kinases in seed development and germination. These SnRK2 proteins were mainly expressed in the nucleus during seed development and germination. The triple mutant (srk2d srk2e srk2i) was sensitive to desiccation and showed severe growth defects during seed development. It exhibited a loss of dormancy and elevated seed ABA content relative to wild-type plants. The severity of these phenotypes was far stronger than that of any single or double SRK2D, SRK2E and SRK2I mutants, including the srk2d srk2i mutant. The triple mutant had greatly reduced phosphorylation activity in in-gel kinase experiments using basic leucine zipper (bZIP) transcription factors including ABI5. Microarray experiments revealed that 48 and 30% of the down-regulated genes in abi5 and abi3 seeds were suppressed in the triple mutant seeds, respectively. Moreover, disruption of the three protein kinases induced global changes in the up-regulation of ABA-repressive gene expression, as well as the down-regulation of ABA-inducible gene expression. These alterations in gene expression result in a loss of dormancy and severe growth defects during seed development. Collectively, these results indicate that SRK2D, SRK2E and SRK2I protein kinases involved in ABA signaling are essential for the control of seed development and dormancy through the extensive control of gene expression.

環境改善施策を評価する場合, 施策による環境改善効果と市場経済への影響の両者を把握する必要がある. 近年, 水環境改善施策の環境評価はGISを用いて詳細に行なわれており, これに合わせた詳細な経済評価が求められている. 筆者らはこれまでに流域GISを援用した流域環境評価モデルと流域経済評価モデルを統合した総合環境評価モデルを構築してきた. 本研究ではこのモデルを用いて, 長良川流域を対象に農業施肥の削減と森林管理の強化という水環境改善施策に対する総合評価を行い, 環境改善による便益や市場経済便益 (不便益) を算出し, 施策実施地域, 施策対象業種, 上下流間の便益帰着構造などを分析することにより.効率的な水環境改善施策に向けた政策的示唆を行う.

Phosphatidic acid (PA) functions as a lipid signaling molecule in plants. Physiological analysis showed that PA triggers early signal transduction events that lead to responses to abscisic acid (ABA) during seed germination. We measured PA production during seed germination and found increased PA levels during early germination. To investigate the role of PA during seed germination, we focused on the PA catabolic enzyme lipid phosphate phosphatase (LPP). LPP catalyzes the conversion of PA to diacylglycerol (DAG). There are 4 LPP genes in the Arabidopsis genome. Among them, AtLPP2 and AtLPP3 are expressed during seed germination. Two AtLPP2 T-DNA insertional mutants (lpp2-1 and lpp2-2) showed hypersensitivity to ABA and significant PA accumulation during germination. Furthermore, double-mutant analysis showed that ABA-insensitive 4 (ABI4) is epistatic to AtLPP2 but ABA-insensitive 3 (ABI3) is not. These results suggest that PA is involved in ABA signaling and that AtLPP2 functions as a negative regulator upstream of ABI4, which encodes an AP2-type transcription factor, in ABA signaling during germination.

Annual changes in the temperature/precipitation and a runoff process in the forest watershed were investigated from the statistical analysis of various time series records. The climate change has not appeared in only a temperature increase but also changes in the precipitation pattern such as the extension of nonprecipitation period, the shortening of precipitation duration and the cyclic interval of heavy rain, and so on. Such climate change become causes of the shortening of precipitation-runoff response, the decrease of the amount of effective precipitation, and the increase of the sediment yields through the increase of evapotranspitation and the decrease of the soil moisture.

A new index was proposed to evaluate the environmental condition in the watershed. Since this index was defined as the statistical variance of the amount of mass flow in the runoff process, it included effects of the continuity on the mass flow/circulation system and the various human activities. As the results of the investigating the definition of the index, it was found that spatial scales of non-point source such as cultivated lands and the runoff length where the natural purification was fully exhibited became important to the environmental assessment.

Phospholipid metabolism is involved in plant responses to drought and salinity stress. To investigate the role of phospholipase D (PLD) and its product phosphatidic acid (PtdOH) in stress signalling, we isolated a novel PLD cDNA, designated AtPLDdelta, by screening a cDNA library prepared from dehydrated Arabidopsis thaliana. The AtPLDdelta protein, of 868 amino acids, has a putative catalytic domain and a C2 domain that is involved in Ca2+/phospholipid binding. The AtPLDdelta mRNA accumulated in response to dehydration and high salt stress. Histochemical analysis showed that the AtPLDdelta gene is strongly expressed in the vascular tissues of cotyledons and leaves under dehydration stress conditions. Under normal growth conditions, AtPLDdelta was expressed in roots, leaves, stems and flowers but not in siliques. We showed that dehydration stimulates the accumulation of PtdOH. The accumulation of PtdOH in response to dehydration was significantly suppressed in AtPLDdelta antisense transgenic plants. These results suggest that AtPLDdelta may be involved in PtdOH accumulation in the dehydration stress response.

In standard P/M (Press-Sinter) technique, it is difficult to make under-cut shape parts. In this development, the possibility of under-cut forming by a CNC press was investigated in the basic experiment. And an under-cut shape sprocket of whole circumference was made by the CNC press and the newly designed die and tools. This paper describes the features of the CNC press and technical issues encountered in the production of the sprocket of the under-cut shape. These are as follows: 1) Elimination of cracks and burrs in compaction, 2) Achievement of an even density throughout the complex part, 3) Keeping the dimensional tolerance and 4) Prevention of breakage of the die and tools. These were resolved by using newly designed die and tools. These are described in detail.

An Arabidopsis thaliana RD20 cDNA, which was isolated as one of drought-inducible genes, encodes a putative protein with a conserved EF-hand Ca2+-binding domain. The recombinant RD20 protein was shown to bind Ca2+. The transcription of RD20 gene was induced not only by drought but also by ABA and high salinity.

In modern higher power, compact and light weight automotive engines, bearing load is increasing and housing stiffness is decreasing. Consequently, the bearing performance is significantly influenced by elastic deformations of both the housing and the bearing. The effects of changing both the housing stiffness and the bearing dimension on bearing performance were studied by using the elastohydrodynamic lubrication theory on connecting rod bearings for diesel engines. [S0742-4787(11)00201-3]

In recent high power, compact and light weight automotive engines, the bearing load has been increasing and housing stiffness has been decreasing. As a result, the bearing performance is greatly influenced by elastic deformations of both the housing and the bearing. The effects of changing both the housing stiffness and the bearing length on bearing performance were studied by using the elastohydrodynamic lubrication theory on connecting rod bearings for diesel engines.

Congenital myasthenic syndromes are caused by mutations in molecules expressed at the neuromuscular junction. Collagen Q (ColQ) makes a triple helical structure and anchors the catalytic subunit of acetylcholinesterase (AChE) to the synaptic basal lamina in the form of asymmetric AChE. Mutations in the collagen Q gene (COLQ) cause endplate AChE deficiency. As an initial step to develop a novel therapeutic strategy for endplate acetylcholinesterase deficiency, we expressed AChE species in cultured cells using retrovirus and adeno-associated virus (AAV). The retroviral vectors carried human ACHE and COLQ either in a single construct (EF1alpha-ACHE-IRES-COLQ) or in two separate constructs (EF1alpha-ACHE and EF1alpha-COLQ). We produced high-titer retroviruses using the PLAT-E retrovirus packaging cells. We also confirmed expression of asymmetric AChE in the PLAT-E cells. We infected NIH3T3 and confirmed expression of the transgenes by RT-PCR. The AAV vector carried human COLQ-IRES-EGFP downstream of the CMV promoter (pAAV-CMV-COLQ-IRES-EGFP). We produced recombinant AAV using HEK293 cells carrying pDF6 encoding the AAV6 capsid gene. We infected AAVHT1080 cells and confirmed expression of COLQ by RT-PCR and EGFP by flow cytometry. We are currently trying to achieve further higher expression levels of transgenes in cultured cells to make the current strategy applicable to an animal model.

In plant cells, plastid DNA is transcribed by at least two types of RNA polymerase, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is homologous to eubacterial transcription machinery, but its regulatory subunit, sigma (sigma) factor, is not encoded on the plastid DNA. We previously cloned the three nuclear-encoded sigma factor genes from Arabidopsis thaliana and designated them as sigA, sigB, and sigC. By means of RFLP mapping, sigA and sigB were mapped on chromosome I and sigC on the chromosome III. Based on comparison of the genomic structure of the three sig genes, intron sites in the 3' half of the genes were shown to be identical between sigB and sigC but divergent in sigA, consistent with the phylogenetic relevance of the three gene products. A transient expression assay of GFP fusions in Arabidopsis protoplasts showed that the N-termini of all three sig gene products functioned as chloroplast-targeting signals. We also constructed transgenic Arabidopsis lines harboring the sigA-promoter or the sigB-promoter uidA fusion. Both the sigA- and sigB-promoters were similarly activated at cotyledons, hypocotyls, rosette leaves, cauline leaves, sepals, and siliques but not at roots, seeds, or other flower organs. In addition, the two promoters were repeatedly activated in young seedlings under continuous light, possibly in an oscillated fashion.

Phosphatidylinositol-4-phosphate 5-kinase (PIP5K) phosphorylates phosphatidylinositol-4-phosphate to produce phosphatidylinositol-4,5-bisphosphate as a precursor of two second messengers, inositol-1,4,5-triphosphate and diacylglycerol, and as a regulator of many cellular proteins involved in signal transduction and cytoskeletal organization. Despite PIP5K playing such an essential role in a number of physiological processes, much still remains to be made clear about its association with plants. Searching the Arabidopsis expression sequence tag database against already known yeast and mammalian PIP5K cDNAs, we identified two clones which partly encode the same Arabidopsis PIP5K and isolated a corresponding full-length cDNA encoding a protein that we designated AtPIP5K1. Recombinant AtPIP5K1 expressed in Escherichia coli possessed a PIP5K activity in vitro. Due to some structural and biochemical differences, AtPIP5K1 was not categorized as either a type I or type II PIP5K. The expression of the AtPIP5K1 mRNA was induced rapidly by treating Arabidopsis plants with drought, salt and abscisic acid, which suggests that AtPIP5K11 is involved in water-stress signal transduction. These data give evidence for a close link between phosphoinositide signaling cascades and water-stress responses in plants.

Plants respond and adapt to environmental changes including drought, high salinity and low temperature. abscisic acid (ABA) plays important roles in these stress responses. A number of plant genes are induced by water stress, such as drought, high salinity and low temperature, and are thought to function in the stress tolerance and responses of the plant. At least four signal transduction pathways control these genes inArabidopsis thaliana: two are ABA-dependent, and two are ABA-independent. Acis-acting element named DRE (Dehydration Responsive Element) is involved in one of the ABA-independent signal transduction pathways, and its DNA binding proteins have been characterized. Drought- and ABA-inducible MYC and MYB homologues are involved in ABA-responsive gene expression inarabidopsis. Roles of thesecis andtrans-acting factors in water stress responses are discussed. In addition, a number of genes for protein kinases, enzymes involved in phosphatidyl inositol metabolism (PI turnover) and transcription factors are also induced by water stress, and thought to be involved in the stress signal transduction cascades. Possible signaling processes in water stress response are discussed.

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 CDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.

The isolation and characterization is reported of a cDNA for delta 1-pyrroline-5-carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana. Southern blot analysis suggested that only one copy of the corresponding gene (AtP5CS) is present in A. thaliana. The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia. Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana. A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.

Pulmonary embolism is a serious, life-threatening condition and most commonly derives from deep vein thrombosis of the lower extremities. Once deep vein thrombosis (DVT) reaches a proximal vein (i.e., popliteal vein or higher), pulmonary embolism reportedly occurs in up to 50% of patients.Case Presentation. We report on an inpatient with major depressive disorder in a catatonic state in whom an asymptomatic proximal deep vein thrombosis of 11 × 70 mm was detected through routine screening, using doppler ultrasound scanning. Anticoagulant therapy was immediately started and continued for three months, which resulted in resolution of the deep vein thrombosis.Discussion. To our knowledge, this is the first description of asymptomatic proximal DVT that was detected in a psychiatric inpatient setting. In light of the reported causal relationship between DVT and pulmonary embolism, screening for DVT can be of high clinical value in patients with psychiatric disorders, especially when their physical activity is highly compromised.

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K+ channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

Two cDNA clones, cATCDPK1 and cATCDPK2, encoding Ca(2+)-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51% and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca(2+)-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca2+ for activation.

A polyethylene cylindrical rod with lamellar structure is loaded with tension-torsion combined stress. The orientation behaviour of the crystallites is evaluated by an improved orientation distribution function. The function assumes affine deformation and that the orthogonal relation of the three crystallographic axes is maintained during deformation. The improved function is achieved by assuming that slippage deformation occurs in a crystallite block. For cases in which twist is strong and samples are highly deformed, the orientation behaviour is interpreted by taking into account the slippage deformation of the (110) plane. This, with the assumption of spiral orientation of the chain axis and rotation of the a- and b-axes around the c-axis, results in good agreement between calculation and experimental results.

A tension-torsion combined stress was loaded onto cylindrical rods of polyethylene to investigate the deformation mechanism of the crystalline phase under non-uniform stress. On applying the combined stress, a neck occurred at the centre of the sample and extended in the axial direction. After necking occurred, the twist deformation generated compressive stress in the radial direction, which suppressed the formation of micro-voids. Wide-angle X-ray diffraction revealed the following facts. The c axis, i.e. the crystal molecular chain axis, deviates from the axial direction owing to the combined loading of the tensile force and the shear force. The b axis, which is the long axis of the initial lamellae, orients selectively in the radial direction in the non-uniform stress state in which the shear stress increases in proportion to the distance from the centre of the cylindrical rod.

Ca2+ is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca2+-permeable channels or transporters involved in Ca2+ uptake in roots are largely unknown. We recently identified a candidate for the Ca2+-permeable mechanosensitive channel in Arabidopsis (Arabidopsis thaliana), named MCA1. Here, we investigated the only paralog of MCA1 in Arabidopsis, MCA2. cDNA of MCA2 complemented a Ca2+ uptake deficiency in yeast cells lacking a Ca2+ channel composed of Mid1 and Cch1. Reverse transcription polymerase chain reaction analysis indicated that MCA2 was expressed in leaves, flowers, roots, siliques, and stems, and histochemical observation showed that an MCA2 promoter::GUS fusion reporter gene was universally expressed in 10-d-old seedlings with some exceptions: it was relatively highly expressed in vascular tissues and undetectable in the cap and the elongation zone of the primary root. mca2-null plants were normal in growth and morphology. In addition, the primary root of mca2-null seedlings was able to normally sense the hardness of agar medium, unlike that of mca1-null or mca1-null mca2-null seedlings, as revealed by the two-phase agar method. Ca2+ uptake activity was lower in the roots of mca2-null plants than those of wild-type plants. Finally, growth of mca1-null mca2-null plants was more retarded at a high concentration of Mg2+ added to medium compared with that of mca1-null and mca2-null single mutants and wild-type plants. These results suggest that the MCA2 protein has a distinct role in Ca2+ uptake in roots and an overlapping role with MCA1 in plant growth.

Three-dimensional finite element (3-D FEM) analysis has been performed on single-edge cracked low density polyethylene to investigate the fracture mechanics for the stationary and growing crack. Input of the mechanical behaviour to the FEM calculation is derived from the uncracked specimen. It is found that, without referring to either the plastic flow theory or to the plastic deformation theory, the 3-D FEM analysis predicts the load-displacement curve of the cracked samples, the crack extension to the applied stress curve and the development of the plastic zone to acceptable agreement with the experimental results. Both the stress and strain at the crack tip are three-dimensional while those remote from the crack tip are close to the two-dimensionsl state.

The neck structure formed in polyethylene cylindrical rods under a tension-torsion combined stress was analyzed using wide-angle X-ray diffraction (WAXD), small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). The volume change of microvoids in the neck was investigated suing DSC and density measurements.At the start of a neck, the crystal a-axis is oriented in the direction that is different from the equatorial direction. At the end of the neck, a fibrillar structure is formed. Bundles of fibers are oriented in the direction that is different from the axial direction due to the resultant force of the combined tensile and shear forces, which result sin a spiral orientation of the polymer chains. Microvoids are created during the plastic deformation as a fibrillar structure is formed. However, the volume of microvoids is reduced in the cold drawing region. As a result, it is suggested that the volume of microvoids created among the bundles of fibers is reduced with further twisting.

The yield behavior of Polyetheretherketone (PEEK) under non-uniform stress state was investigated using cylindrical rods loaded with tension-torsion combined stress. Along with the recording of tensile force and torsional torque, the change of sample shape was photographed at different stages of loading. The starting point of plastic deformation was discussed by according to the Nadai's equation. The load-unload cyclic tests under the combined stress were conducted in order to examine the generation of permanent strain. As a result, permanent strain in sample axis direction was generated after the maximum point in the torque-time curve, and this point is considered as the limit for use.

Responses to water stress are thought to be mediated by transcriptional regulation of gene expression via reversible protein phosphorylation events. Previously, we reported that bZIP (basic-domain leucine zipper)-type AREB/ABF (ABA-responsive element-binding protein/factor) transcription factors are involved in ABA signaling under water stress conditions in Arabidopsis. The AREB1 protein is phosphorylated in vitro by ABA-activated SNF1-related protein kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). Consistent with this, we now show that SRK2D/E/I and AREB1 co-localize and interact in nuclei in planta. Our results show that unlike srk2d, srk2e and srk2i single and double mutants, srk2d srk2e srk2i (srk2d/e/i) triple mutants exhibit greatly reduced tolerance to drought stress and highly enhanced insensitivity to ABA. Under water stress conditions, ABA- and water stress-dependent gene expression, including that of transcription factors, is globally and drastically impaired, and jasmonic acid (JA)-responsive and flowering genes are up-regulated in srk2d/e/i triple mutants, but not in other single and double mutants. The down-regulated genes in srk2d/e/i and areb/abf triple mutants largely overlap in ABA-dependent expression, supporting the view that SRK2D/E/I regulate AREB/ABFs in ABA signaling in response to water stress. Almost all dehydration-responsive LEA (late embryogenesis abundant) protein genes and group-A PP2C (protein phosphatase 2C) genes are strongly down-regulated in the srk2d/e/i triple mutants. Further, our data show that these group-A PP2Cs, such as HAI1 and ABI1, interact with SRK2D. Together, our results indicate that SRK2D/E/I function as main positive regulators, and suggest that ABA signaling is controlled by the dual modulation of SRK2D/E/I and group-A PP2Cs.

Congenital myasthenic syndromes are caused by mutations in molecules expressed at the neuromuscular junction. Collagen Q (ColQ) makes a triple helical structure and anchors the catalytic subunit of acetylcholinesterase (AChE) to the synaptic basal lamina in the form of asymmetric AChE. Mutations in the collagen Q gene (COLQ) cause endplate AChE deficiency. As an initial step to develop a novel therapeutic strategy for endplate acetylcholinesterase deficiency, we expressed AChE species in cultured cells using retrovirus and adeno-associated virus (AAV). The retroviral vectors carried human ACHE and COLQ either in a single construct (EF1alpha-ACHE-IRES-COLQ) or in two separate constructs (EF1alpha-ACHE and EF1alpha-COLQ). We produced high-titer retroviruses using the PLAT-E retrovirus packaging cells. We also confirmed expression of asymmetric AChE in the PLAT-E cells. We infected NIH3T3 and confirmed expression of the transgenes by RT-PCR. The AAV vector carried human COLQ-IRES-EGFP downstream of the CMV promoter (pAAV-CMV-COLQ-IRES-EGFP). We produced recombinant AAV using HEK293 cells carrying pDF6 encoding the AAV6 capsid gene. We infected AAVHT1080 cells and confirmed expression of COLQ by RT-PCR and EGFP by flow cytometry. We are currently trying to achieve further higher expression levels of transgenes in cultured cells to make the current strategy applicable to an animal model.

Plants can sense and respond to mechanical stimuli, like animals. An early mechanism of mechanosensing and response is speculated to be governed by as-yet-unidentified sensory complexes containing a Ca 2+ -permeable, stretch-activated (SA) channel. However, the components or regulators of such complexes are poorly understood at the molecular level in plants. Here, we report the molecular identification of a plasma membrane protein (designated Mca1) that correlates Ca 2+ influx with mechanosensing in Arabidopsis thaliana . MCA1 cDNA was cloned by the functional complementation of lethality of a yeast mid1 mutant lacking a putative Ca 2+ -permeable SA channel component. Mca1 was localized to the yeast plasma membrane as an integral membrane protein and mediated Ca 2+ influx. Mca1 also increased [Ca 2+ ] cyt upon plasma membrane distortion in Arabidopsis . The growth of MCA1 -overexpressing plants was impaired in a high-calcium but not a low-calcium medium. The primary roots of mca1 -null plants failed to penetrate a harder agar medium from a softer one. These observations demonstrate that Mca1 plays a crucial role in a Ca 2+ -permeable SA channel system that leads to mechanosensing in Arabidopsis . We anticipate our findings to be a starting point for a deeper understanding of the molecular mechanisms of mechanotransduction in plants.

Phosphatidic acid (PA) functions as a lipid signaling molecule in plants. Physiological analysis showed that PA triggers early signal transduction events that lead to responses to abscisic acid (ABA) during seed germination. We measured PA production during seed germination and found increased PA levels during early germination. To investigate the role of PA during seed germination, we focused on the PA catabolic enzyme lipid phosphate phosphatase (LPP). LPP catalyzes the conversion of PA to diacylglycerol (DAG). There are 4 LPP genes in the Arabidopsis genome. Among them, AtLPP2 and AtLPP3 are expressed during seed germination. Two AtLPP2 T-DNA insertional mutants (lpp2-1 and lpp2-2) showed hypersensitivity to ABA and significant PA accumulation during germination. Furthermore, double-mutant analysis showed that ABA-insensitive 4 (ABI4) is epistatic to AtLPP2 but ABA-insensitive 3 (ABI3) is not. These results suggest that PA is involved in ABA signaling and that AtLPP2 functions as a negative regulator upstream of ABI4, which encodes an AP2-type transcription factor, in ABA signaling during germination.

The focus of this study was to accurately measure the oil film thickness across the width of a connecting rod bearing operating under load. A new experimental method using Laser Induced Fluorescence (LIF) was developed for this purpose. The oil film thickness setup was first calibrated under static load and the accuracy of the system was successfully confirmed. The experiment indicated that the oil film thickness near the outer edges of the bearing was thinner than at the center in all tested conditions. This phenomenon has not been verified experimentally until now. The experimental results were confirmed by Elastohydrodynamic Lubrication (EHL). This study indicates that the experimental method is useful to prove the calculation.