Generation and Characterization of Highly Constitutive Active Histamine H3 Receptors

ABSTRACT Constitutive activation of G-protein-coupled receptors is a well recognized phenomenon, and G-protein-coupled receptor antagonists have been found to possess inverse agonist activity. Constitutive activation of histamine H3 receptor is recently documented in in vivo as well as in recombinant receptor systems in vitro. Several H3 antagonists have been shown to act as inverse agonists and such profiles of H3 antagonists have been implicated in their pharmacological functions. Here we report the construction and characterization of a highly constitutive active H3 receptor (MT6), in which the 357 alanine residue was converted to lysine (A357K). We generated a series of mutated H3 receptors and their functions were examined in human embryonic kidney (HEK) 293 cells. Among them, induced mutation at the amino acid 357 position (A357K) showed a dramatically enhanced response to thioperamide-induced cAMP accumulation compared with the cells expressing wildtype (WT) H3 receptors, suggesting that the mutation rendered receptors to high constitutive activity. We further characterized by ligand binding assays using membrane fractions, and K i values of imetit (agonist) and proxyfan (partial agonist) against the MT6 receptors were lower compared with those observed in WT H3 receptors. In contrast, H3 antagonists (thioperamide, ciproxifan, and GT2016) with inverse agonism displayed increased K i values against the MT6 receptors (2.5-to 5.8-fold), demonstrating more a prominent effect of inverse agonists to the constitutive active receptor. Taken together, these data suggested that A357K mutation in the H3 receptor increased the population of active state receptors that preferably binds to agonists than inverse agonists, which could be termed as a constitutively active mutant of H3 receptor

ホッカイドウ トウブ・アカンコ シュウヘン ニ オケル エゾマツ ジャクレイ ゾウリンボク ノ セイイク ト カンキョウ

北海道の天然林におけるエゾマツ資源の減少を背景として，人工造林によるエゾマツ資源の回復が期待されている。エゾマツ造林の成功率向上に資するため，北海道東部の阿寒湖周辺の2～18年生のエゾマツ若齢造林地約30か所において，造林木の生育状況と立地環境を調査し，造林木の生存と樹高成長に及ぼす各種の立地環境要因の影響を解析した。造林木の生存率は，2齢級の造林地で40%を下回る調査地がいくつか見られたものの全体的には比較的高く，生存率50%以上の調査地が全調査地数の87%を占めた。樹高成長に関しては，林齢18年生までで平均樹高が3mを超える調査地はなく，既往の報告と比べると全般的に小さかった。エゾマツ造林木の生存と成長に及ぼす立地環境要因の影響を一般化線形モデル（GLM）によって解析した結果，造林木の生存率には，標高，斜面傾斜，斜面方位，散乱光透過率（DSF），上木の針葉樹比率，およびDSFと上木の針葉樹比率の交互作用が影響を及ぼしていることが示唆された。一方，造林木の平均樹高には，林齢，DSF, 上木の針葉樹比率が影響を及ぼしていることが示唆された。GLMによる解析結果を用いて造林木の生存率を散乱光透過率DSFとの関係で予測したところ，生存率はDSFが小さいほど低くなり，とくにDSFが約35%より小さい領域では生存率に及ぼす針葉樹比率の影響が強く現れ，針葉樹比率が高いほどDSFの低下にともなう生存率の低下が顕著になると予測された。同様に，造林木の平均樹高をDSFとの関係で予測したところ，平均樹高はDSFが大きいほど高く，上木の針葉樹比率が低いほど高くなった。これらの結果から，DSFが約35%を下回るような暗い天然林内でとくに上木の針葉樹比率が高い場所にエゾマツを樹下植栽する場合には，DSF35％以上を確保するために少なくとも900m2程度のギャップを作出するような更新伐が必要と考えられた。Timber resources of Ezo spruce, Picea jezoensis (Siebold et Zucc.) Carrière, a coniferous tree species representative of natural forests in Hokkaido, northern Japan, have declined due to intensive logging activities and the difficulty of natural regeneration in this species. To obtain technical information for the improvement of artificial Ezo spruce regeneration, we investigated the growth and survival of planted young trees in approximately 30 stands in the forest surrounding Lake Akan, eastern Hokkaido, Japan. Most of the investigated trees were grown beneath the canopy of a natural mixed forest comprising evergreen conifers and deciduous broadleaf trees, except for a few open sites. The ages of the planted trees, which we defined as the number of years after plantation, ranged from 2 to 18 years. We also measured various environmental factors such as relative light intensity (diffuse site factor, DSF) and the proportion of evergreen conifers to the total basal area of the surrounding canopy trees. Survival rates were relatively high among the planted trees, at ≥50% in 87% of all stands. However, in several stands, survival rates were ＜40% in the 6-10 years age class. Height increases were smaller than those reported in previous studies ; the average height of planted trees did not exceed 3 m in any stands aged ＜18 years. Generalized linear model (GLM) analysis showed that tree survival was affected by altitude, slope inclination, slope aspect, DSF, the proportion of conifers in the canopy layer, and the interaction between DSF and the proportion of conifers in the canopy layer. This analysis also showed that average height was influenced by age, DSF, and the proportion of conifers in the canopy layer. We applied these results to predict survival rates and average heights of trees as a function of DSF and the proportion of conifers in the canopy layer. The predicted survival rates decreased as DSF decreased, and the proportion of conifers in the canopy layer enhanced the decline in survival rates at DSF values ＜ca. 35%. The GLM prediction also showed that average height would be higher in stands with higher DSF values and lower proportions of conifers in the canopy layer. Based on these results and the relationship between DSF and gap size, we conclude that a canopy gap of at least 900 m2 may be desirable when Ezo spruce trees are planted beneath the canopy, especially in mixed stands where the proportion of evergreen conifers in the canopy layer is high and DSF is ＜ca. 35%

Microstructure Investigation of Polymer Electrolyte Fuel Cell Catalyst Layers Containing Perfluorosulfonated Ionomer

Perfluorosulfonated ionomers are the most successful ion-exchange membranes at an industrial scale. One recent, cutting-edge application of perfluorosulfonated ionomers is in polymer electrolyte fuel cells (PEFCs). In PEFCs, the ionomers are used as a component of the catalyst layer (CL) in addition to functioning as a proton-exchange membrane. In this study, the microstructures in the CLs of PEFCs were characterized by combined synchrotron X-ray scattering and transmission electron microscopy (TEM) analyses. The CL comprised a catalyst, a support, and an ionomer. Fractal dimensional analysis of the combined ultrasmall- and small-angle X-ray scattering profiles indicated that the carbon-black-supported Pt catalyst (Pt/CB) surface was covered with the ionomer in the CL. Anomalous X-ray scattering revealed that the Pt catalyst nanoparticles on the carbon surfaces were aggregated in the CLs. These findings are consistent with the ionomer/catalyst microstructures and ionomer coverage on the Pt/CB surface obtained from TEM observations

Distinct cerebellar engrams in short-term and long-term motor learning

Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF-PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF-PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF-PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-term adaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF-PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF-PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF-PC synapses and the elimination of these synapses are in vivo engrams in short- and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation

Lipoprotein lipase does not increase significantly in the postprandial plasma.

BackgroundPrevious reports have shown that lipoprotein lipase (LPL) activity significantly increases in the postprandial plasma associated with the increase of TG-rich lipoproteins. Therefore, we have reexamined those relationships using newly developed LPL assay with the different kinds of food intake.MethodsStandard meal (n=81), 50g of fat (n=54), 75g of glucose (n=25) and cookie (25g fat and 75g carbohydrate fat) (n=28) were administered in generally healthy volunteers. Plasma LPL, HTGL and TC, TG, LDL-C, HDL-C, RLP-C and RLP-TG were determined at subsequent withdrawal after the food intake.ResultsPlasma TG, RLP-C and RLP-TG were significantly increased at 8PM (2h after dinner of standard meal) compared with 8AM before breakfast within the same day. Also those parameters were significantly increased in 2-6h after fat load. However, the concentrations and activities of LPL and HTGL did not significantly increase in association with an increase in the TG and remnant lipoproteins. Also LPL concentration did not significantly increase after glucose and "cookie test" within 4h.ConclusionNo significant increase of LPL activity was found at CM and VLDL overload after different kinds of food intake when reexamined by newly developed assay for LPL activity and concentration

IL-17A-induced hypercontractility and its signal transduction in cultured human colonic SMCs.

<p>(<b>A</b>) Contractility assay of IL-17A-treated human SMCs. SMCs were cultured with IL-17A and IL-4 for 5 days and contractility was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092960#pone-0092960-g006" target="_blank">Figure? 6A</a>. (<b>B</b>) Immunoblot analysis of p-MLC and quantitation of band intensity in samples of colonic SMCs treated with or without IL-17A, IL-1β or IL-4 (n = 6). The cells were treated with 10⁻¹¹ M CCh for 3 min. The ratio of p-MLC to total MLC was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092960#s2" target="_blank">Materials and Methods</a> and normalized to untreated samples. N refers to the number of experiments. Upper panels are representative images. (<b>C</b>) Relative intensity of NFκB p65 immunosignal accumulated in the nucleus was measured in cultured colonic SMCs after 30 min treatment with PBS, IL-17A or IL-1β in the absence or presence of anti-IL-17RC antibody or siRNAs to control and IL-17RC. (<b>D</b>) The effect of IL-17A and IL-1β on RGS 4 activity in human SMCs on day 4 was assessed (n = 4). RGS4 activity was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092960#s2" target="_blank">Materials and Methods</a>. (<b>E</b>) Effect of anti-IL-17RC antibody, SN50 and IκBζ siRNA on IL-17A-induced contractility in human SMCs. Contractility was measured on day 4 (n = 3–6) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092960#pone-0092960-g006" target="_blank">Fig.6A</a>. (<b>F</b>) The effects of MAPK inhibitors on IL-17A- and anisomycin-induced hypercontractility on day 4 (n = 3–6). The left and right panels show the effects MAPK inhibitors on IL-17A-, and anisomycin-induced contractility, respectively. Contractility was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092960#pone-0092960-g006" target="_blank">Fig. 6A</a> (n = 3). Numerical data represent means ± s.e.m. *P<0.05, **P<0.01, Student's t-test under the closed testing procedure for multiple comparison (<b>A–F</b>).</p