Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).     

Molecular phylogeny and taxonomic implications of Asarum (Aristolochiaceae) based on ITS and matK sequences

The genus Asarum (Aristolochiaceae) encompasses approximately 120 species from five sections. Taxonomic controversies concerning the genus Asarum and/or its intrageneric classification remain unresolved. In particular, sect. Heterotropa accounts for a large percentage of the genus (80 of 120 species) and is well diverged in the Sino–Japanese Forest subkingdom. Reconstruction of Heterotropa phylogeny and estimation of its divergence times would provide significant insight into the process of species diversity in the Sino–Japanese floristic region. This study encompassed 106 operational taxonomic units (OTUs), and phylogenetic analyses were conducted based on internal transcribed spacer (ITS) and matK sequences. Although the matK sequences provided informative results solely for section Geotaenium, phylogenetic trees based on ITS regions yielded a clear result for several sections. Three sections, Asarum, Geotaenium and Asiasarum, were supported as robust monophyletic groups, whereas Heterotropa had low support. Sect. Hexastylis was revealed to be polyphyletic, suggesting taxonomic reconstruction would be needed. Sect. Heterotropa comprises two clades, which correspond to species distribution ranges: mainland China and the island arc from Taiwan to mainland Japan via the Ryukyu Islands. It is notable that the common ancestry of the latter clade in the eastern Asian islands was highly supported, suggesting that the present species diversity of Heterotropa was initially caused by allopatric range fragmentation in East Asia.     

Amelioration of diabetic nephropathy by oral administration of d-α-tocopherol and its mechanisms

Diabetic nephropathy (DN) is a diabetic vascular complication, and abnormal protein kinase C (PKC) activation from increased diacylglycerol (DG) production in diabetic hyperglycemia is one of the causes of DN. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. In other words, DGK can attenuate PKC activity by reducing the amount of DG. Recently, we reported that intraperitoneally administered d-α-tocopherol (vitamin E, αToc) induces an amelioration of DN in vivo through the activation of DGKα and the prevention of podocyte loss. However, the effect of the oral administration of αToc on DN in mice remains unknown. Here, we evaluated the effect of oral administration of αToc on DN and its molecular mechanism using streptozocin-induced diabetic mice. Consequently, the oral administration of αToc significantly ameliorated the symptoms of DN by preventing the loss of podocytes, and it was revealed that the inhibition of PKC?activity was involved in this amelioration.     

Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid,a histone deacetylase inhibitor

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.     

Prey pursuit strategy of Japanese horseshoe bats during an in-flight target-selection task

The prey pursuit behavior of Japanese horseshoe bats (Rhinolophus ferrumequinum nippon) was investigated by tasking bats during flight with choosing between two tethered fluttering moths. Echolocation pulses were recorded using a telemetry microphone mounted on the bat combined with a 17-channel horizontal microphone array to measure pulse directions. Flight paths of the bat and moths were monitored using two high-speed video cameras. Acoustical measurements of returning echoes from fluttering moths were first collected using an ultrasonic loudspeaker, turning the head direction of the moth relative to the loudspeaker from 0° (front) to 180° (back) in the horizontal plane. The amount of acoustical glints caused by moth fluttering varied with the sound direction, reaching a maximum at 70°–100° in the horizontal plane. In the flight experiment, moths chosen by the bat fluttered within or moved across these angles relative to the bat’s pulse direction, which would cause maximum dynamic changes in the frequency and amplitude of acoustical glints during flight. These results suggest that echoes with acoustical glints containing the strongest frequency and amplitude modulations appear to attract bats for prey selection.     

Thrombopoietin receptor-based protein–protein interaction screening (THROPPIS)

As protein–protein interactions (PPIs) are involved in many cellular events, development of mammalian cytosolic PPI detection systems is important for drug discovery as well as understanding biological phenomena. We have previously reported a c-kit-based PPI screening (KIPPIS) system, in which proteins of interest were fused with a receptor tyrosine kinase c-kit, leading to intracellular PPI-dependent cell growth. However, it has not been investigated whether PPI can be detected using other receptors. In this study, we employed a thrombopoietin receptor, which belongs to the Type I cytokine receptor family, to develop a thrombopoietin receptor-based PPI screening (THROPPIS) system. To improve the sensitivity of THROPPIS, we examined two strategies of (i) localization of the chimeric receptors on the cell membrane, and (ii) addition of a helper module to the chimeric receptors. Intriguingly, the nonlocalized chimeric receptor showed the best performance of THROPPIS. Furthermore, the addition of the helper module dramatically improved the detection sensitivity. In total, 5 peptide–domain interactions were detected successfully, demonstrating the versatility of THROPPIS. In addition, a peptide–domain interaction was detected even when insulin receptor or epidermal growth factor receptor was used as a signaling domain, demonstrating that this PPI detection system can be extended to other receptors.     

Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure.     

Photoelectrochemistry of metalloporphyrin-modified GaP semiconductors

Photosynthesis Research - Photoelectrosynthetic materials provide a bioinspired approach for using the power of the sun to produce fuels and other value-added chemical products. However, there...     

Effects of yokukansan on behavioral and psychological symptoms of vascular dementia: an open-label trial

Previous clinical trials suggest that the traditional Japanese medicine yokukansan has beneficial effects on the behavioral and psychological symptoms of dementia (BPSD). The present study was conducted to elucidate the efficacy of yokukansan on BPSD in patients with vascular dementia. Thirteen Japanese patients (9 men and 4 women) who were diagnosed as having vascular dementia (VaD) according to the diagnostic criteria of NINDS-AIREN were subjected to the open-label clinical trial in which yokukansan (7.5g/day) has been given for 4 weeks. Their mean age was 71.2±6.5 years. The BPSD was evaluated using the Neuropsychiatric Inventory (NPI), cognitive function was evaluated by the Mini-Mental State Examination (MMSE), the activities of daily living was evaluated by Barthel index (BI) and Disability Assessment for Dementia (DAD), and the extrapyramidal signs were evaluated by United Parkinson's Disease Rating Scale (UPDRS). The mean NPI was 33.0±17.3 and 23.6±13.9 for the baseline and after treatment, respectively. It was significantly improved after treatment (p<0.05). In the NPI-subcategories, there was a significant improvement in agitation and disinhibition after the treatment. There was no significant change in MMSE, BI, DAD or UPDRS before and after the treatment. There was no adverse effect during the treatment period. The present results suggest that yokukansan is beneficial for the treatment of BPSD in VaD patients.