Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori.

Nongenomic action of an insect steroid hormone, 20-hydroxyecdysone (20E), has been implicated in several 20E-dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [3H]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1 m NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [3H]PonA (Kd = 17.3 nm, Bmax = 0.82 pmol.mg(-1) protein). Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-dependent developmental events.     

Enhanced Stomatal Conductance by a Spontaneous Arabidopsis Tetraploid,Me-0, Results from Increased Stomatal Size and Greater Stomatal Aperture

The rate of gas exchange in plants is regulated mainly by stomatal size and density. Generally, higher densities of smaller stomata are advantageous for gas exchange; however, it is unclear what the effect of an extraordinary change in stomatal size might have on a plant’s gas-exchange capacity. We investigated the stomatal responses to CO₂ concentration changes among 374 Arabidopsis (Arabidopsis thaliana) ecotypes and discovered that Mechtshausen (Me-0), a natural tetraploid ecotype, has significantly larger stomata and can achieve a high stomatal conductance. We surmised that the cause of the increased stomatal conductance is tetraploidization; however, the stomatal conductance of another tetraploid accession, tetraploid Columbia (Col), was not as high as that in Me-0. One difference between these two accessions was the size of their stomatal apertures. Analyses of abscisic acid sensitivity, ion balance, and gene expression profiles suggested that physiological or genetic factors restrict the stomatal opening in tetraploid Col but not in Me-0. Our results show that Me-0 overcomes the handicap of stomatal opening that is typical for tetraploids and achieves higher stomatal conductance compared with the closely related tetraploid Col on account of larger stomatal apertures. This study provides evidence for whether larger stomatal size in tetraploids of higher plants can improve stomatal conductance.Gas exchange is a vital activity for higher plants that take up atmospheric CO₂ and release oxygen and water vapor through epidermal stomatal pores. Gas exchange affects CO₂ uptake, photosynthesis, and biomass production (Horie et al., 2006; Evans et al., 2009; Tanaka et al., 2014). Stomatal conductance (gs) is used as an indicator of gas-exchange capacity (Franks and Farquhar, 2007). Maximum stomatal conductance (gsmax) is controlled mainly by stomatal size and density, two parameters that change with environmental conditions and are negatively correlated with each other (Franks et al., 2009).Given a constant total stomatal pore area, large stomata are generally disadvantageous for gas exchange compared with smaller stomata, because the greater pore depth in larger stomata increases the distance that gas molecules diffuse through. This increased distance is inversely proportional to g_smax (Franks and Beerling, 2009). The fossil record indicates that ancient plants had small numbers of large stomata when atmospheric CO₂ levels were high, and falling atmospheric [CO₂] induced a decrease in stomatal size and an increase in stomatal density to increase g_s for maximum carbon gain (Franks and Beerling, 2009). The positive relationship between a high g_s and numerous small stomata also holds true among plants living today under various environmental conditions (Woodward et al., 2002; Galmés et al., 2007; Franks et al., 2009). Additionally, the large stomata of several plant species (e.g. Vicia faba and Arabidopsis [Arabidopsis thaliana]) are often not effective for achieving rapid changes in g_s, due to slower solute transport to drive movement caused by their lower membrane surface area-to-volume ratios (Lawson and Blatt, 2014).Stomatal size is strongly and positively correlated with genome size (Beaulieu et al., 2008; Franks et al., 2012; Lomax et al., 2014). Notably, polyploidization causes dramatic increases in nucleus size and stomatal size (Masterson, 1994; Kondorosi et al., 2000). In addition to the negative effects of large stomata on gas exchange (Franks et al., 2009), polyploids may have another disadvantage; del Pozo and Ramirez-Parra (2014) showed that artificially induced tetraploids of Arabidopsis have a reduced stomatal density (stomatal number per unit of leaf area) and a lower stomatal index (stomatal number per epidermal cell number). Moreover, tetraploids of Rangpur lime (Citrus limonia) and Arabidopsis have lower transpiration rates and changes in the expression of genes involved in abscisic acid (ABA), a phytohormone that induces stomatal closure (Allario et al., 2011; del Pozo and Ramirez-Parra, 2014). On the other hand, an increase in the ploidy level of Festuca arundinacea results in an increase in the CO₂-exchange rate (Byrne et al., 1981); hence, polyploids may not necessarily have a reduced gas-exchange capacity.Natural accessions provide a wide range of information about mechanisms for adaptation, regulation, and responses to various environmental conditions (Bouchabke et al., 2008; Brosché et al., 2010). Arabidopsis, which is distributed widely throughout the Northern Hemisphere, has great natural variation in stomatal anatomy (Woodward et al., 2002; Delgado et al., 2011). Recently, we investigated leaf temperature changes in response to [CO₂] in a large number of Arabidopsis ecotypes (374 ecotypes; Takahashi et al., 2015) and identified the Mechtshausen (Me-0) ecotype among ecotypes with low CO₂ responsiveness; Me-0 had a comparatively low leaf temperature, implying a high transpiration rate. In this study, we revealed that Me-0 had a higher g_s than the standard ecotype Columbia (Col), despite having tetraploid-dependent larger stomata. Notably, the g_s of Me-0 was also higher than that of tetraploid Col, which has stomata as large as those of Me-0. This finding resulted from Me-0 having a higher g_s-to-g_smax ratio due to more opened stomata than tetraploid Col. In addition, there were differences in ABA responsiveness, ion homeostasis, and gene expression profiles in guard cells between Me-0 and tetraploid Col, which may influence their stomatal opening. Despite the common trend of smaller stomata with higher gas-exchange capacity, the results with Me-0 confirm the theoretical possibility that larger stomata can also achieve higher stomatal conductance if pore area increases sufficiently.     

Effect of One-Week Salt Restriction on Blood Pressure Variability in Hypertensive Patients with Type 2 Diabetes

Background

Increased short-term blood pressure (BP) variability on 24-hour ambulatory BP monitoring (ABPM) is known to be a risk factor for cardiovascular events. However, very few studies have evaluated the effect of salt restriction on BP variability particularly in hypertensive patients with type 2 diabetes. This study aimed to investigate the effect of salt restriction on systolic BP (SBP) variability.

Methods and Results

10 hypertensive patients with type 2 diabetes and not receiving antihypertensive agents were enrolled in the study. After admission, all patients received a salt-restricted diet and appropriate anti-diabetic treatments and were followed up for 7 consecutive days using ABPM. After the 7-day treatment, the median [interquartile range (IQR)] coefficient of variation (CV) for diurnal SBP variability changed from day 1 to day 7–13.0 [10.8 to 16.8] % to 13.3 [9.1 to 18.9] % (P = 0.959)—and the median [IQR] change between days 1 and 7 was -0.3 [-3.2 to 2.9] %. In addition, CV for BP variability and circadian rhythm of BP varied greatly on a day-by-day basis for 7 days, compared to mean BP values. Interestingly, increased SBP variability was associated with greater day-by-day changes in circadian rhythm of BP.

Conclusions

Salt restriction during 7-day hospitalization led to a -0.3 [-3.2 to 2.9] (median [IQR]) % change from baseline in CV for diurnal SBP variability in 10 hypertensive patients with type 2 diabetes not receiving antihypertensive agents.

Trial Registration

UMIN Clinical Trials Registry UMIN000016243     

Characteristics of ammonia oxidation potentials and ammonia oxidizers in mineral soil under <Emphasis Type="Italic">Salix polaris</Emphasis>–moss vegetation in Ny-Ålesund,Svalbard

Although nitrification is a unique and important process in the nitrogen cycle with respect to ammonium consumption and nitrate production, limited information on this process is available for high-Arctic soils. We elucidated the ammonia oxidation potentials (AOPs) and characteristics of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in mineral soils under climax vegetation, i.e., Salix polaris (polar willow)–moss vegetation, on a coastal hill in Ny-Ålesund, Svalbard. AOPs at 10 °C were determined by incubation with sufficient substrate (2 mM ammonium). The ammonia monooxygenase subunit A (amoA) genes of AOB and AOA were analyzed by using quantitative polymerase chain reaction and pyrosequencing. AOPs ranged from 1.1 to 14.1 ng N g^?1 dry soil h^?1—relatively low but of a similar order to the gross nitrification rates reported in another Svalbard study. AOP was positively correlated with thickness of the moss layer (P < 0.01), soil water content, and ammonium nitrogen content (P < 0.05). The population sizes of both AOB and AOA were not significantly related to AOP or edaphic factors. For AOB-amoA, six major operational taxonomic units (OTUs) were identified, all of which were classified into the Nitrosospira Mount Everest cluster. For AOA-amoA, six major OTUs were also identified, five of which were grouped with sequences from cold environments within clade A of the Nitrososphaera cluster, i.e., species known to have low, or no, AOP. It is, therefore, possible that the AOPs measured at the study site were driven mainly by psychrotolerant AOB.     

DNA methylation errors in cloned mice disappear with advancement of aging

Cloned animals have various health problems. Aberrant DNA methylation is a possible cause of the problems. Restriction landmark genomic scanning (RLGS) that enabled us to analyze more than 1,000 CpG islands simultaneously demonstrated that all cloned newborns had aberrant DNA methylation. To study whether this aberration persists throughout the life of cloned individuals, we examined genome-wide DNA methylation status of newborn (19.5 dpc, n=2), adult (8-11 months old, n=3), and aged (23-27 months old, n=4) cloned mice using kidney cells as representatives. In the adult and aged groups, cloning was repeated using cumulus cells of the adult founder clone of each group as nucleus donor. Two newborn clones had three with aberrantly methylated loci, which is consistent with previous reports that all cloned newborns had DNA methylation aberrations. Interestingly, we could detect only one aberrantly methylated locus in two of the three adult clones in mid-age and none of four senescent clones, indicating that errors in DNA methylation disappear with advancement of animals' aging.     

Phenylimidazole derivatives as specific inhibitors of bacterial enoyl-acyl carrier protein reductase FabK

Bacterial enoyl-acyl carrier protein (ACP) reductases (FabI and FabK) catalyze the final step in each cycle of bacterial fatty acid biosynthesis and are attractive targets for the development of new antibacterial agents. Here, we report the development of novel FabK inhibitors with antibacterial activity against Streptococcus pneumoniae. Based on structure-activity relationship (SAR) studies of our screening hits, we have developed novel phenylimidazole derivatives as potent FabK inhibitors.     

Evidence for new beta1-3 galactosyltransferase activity involved in biosynthesis of unusual N-glycan harboring T-antigen in Apis mellifera

In a previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 70, 2583-2587, 2006), we found that new complex type N-glycans harboring Thomsen-Friedenreich antigen (Galbeta1-3GalNAc) unit occur on royal jelly glycoproteins, suggesting the involvement of a new beta1-3galactosyltransferase in the synthesis of the unusual complex type N-glycans. So far, such beta1-3galactosyltransferase activity, which can transfer galactosyl residues with the beta1-3 linkage to beta1-4 GalNAc residues in N-glycan, has not been found among any eucaryotic cells. But using GalNAc(2)GlcNAc(2)Man(3)GlcNAc(2)-PA as acceptor N-glycan, we detected the beta1-3 galactosyltransferase activity in membrane fraction prepared from honeybee cephalic portions. This result indicates that honeybee expresses a unique beta1-3 galactosyltransferase involved in biosynthesis of the unusual N-glycan containing a tumor related antigen in the hypopharyngeal gland.     

Loop 1 structure of the leishmanial ICAM-L molecule is crucial for parasite binding and infection of host macrophages

The binding of each intercellular adhesive molecule (ICAM) molecule fragment from Leishmania amazonensis (ICAM-L) to host macrophages was investigated using an indirect immunofluorescent sandwich technique, based on the observation that ICAM-L can block the uptake of L. amazonensis on the macrophage surface and all prepared ICAM-L fragments can react with rabbit anti-ICAM-L antiserum. The ICAM-L fragments lacking the loop 1 (LI) structure failed to bind to macrophages, and the disruption of the LI structure by mercaptoethanol led to the failure of binding. The fragments containing the LI structure functioned similarly to ICAM-L, by temporarily retarding host cell growth and cell cycle progression, and inhibiting the Leishmania infection of host macrophages. These results suggest that LI constitutes the main determinant of the ICAM-L molecule in binding to, and infection of, host macrophages.     

Knock down of gamma-glutamylcysteine synthetase in rat causes acetaminophen-induced hepatotoxicity

Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. Gamma-glutamylcysteine synthetase (gamma-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat gamma-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10(11) pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.     

Random PCR‐based genotyping by sequencing technology GRAS‐Di (genotyping by random amplicon sequencing,direct) reveals genetic structure of mangrove fishes

While various technologies for high‐throughput genotyping have been developed for ecological studies, simple methods tolerant to low‐quality DNA samples are still limited. In this study, we tested the availability of a random PCR‐based genotyping‐by‐sequencing technology, genotyping by random amplicon sequencing, direct (GRAS‐Di). We focused on population genetic analysis of estuarine mangrove fishes, including two resident species, the Amboina cardinalfish (Fibramia amboinensis, Bleeker, 1853) and the Duncker's river garfish (Zenarchopterus dunckeri, Mohr, 1926), and a marine migrant, the blacktail snapper (Lutjanus fulvus, Forster, 1801). Collections were from the Ryukyu Islands, southern Japan. PCR amplicons derived from ~130 individuals were pooled and sequenced in a single lane on a HiSeq2500 platform, and an average of three million reads was obtained per individual. Consensus contigs were assembled for each species and used for genotyping of single nucleotide polymorphisms by mapping trimmed reads onto the contigs. After quality filtering steps, 4,000–9,000 putative single nucleotide polymorphisms were detected for each species. Although DNA fragmentation can diminish genotyping performance when analysed on next‐generation sequencing technology, the effect was small. Genetic differentiation and a clear pattern of isolation‐by‐distance was observed in F. amboinensis and Z. dunckeri by means of principal component analysis, F_ST and the admixture analysis. By contrast, L. fulvus comprised a genetically homogeneous population with directional recent gene flow. These genetic differentiation patterns reflect patterns of estuary use through life history. These results showed the power of GRAS‐Di for fine‐grained genetic analysis using field samples, including mangrove fishes.