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Tuberculosis is a major threat to global health and its increased incidence in adolescents as well as onset in the elderly presents a serious problem. One strategy to control tuberculosis involves taking advantage of Bacillus Calmette-Guerin's (BCG) superior effects on childhood tuberculosis. Accordingly, here we aimed to develop a booster vaccine for adults who received the BCG vaccine during early childhood. Therefore, we first devised a system to assess the efficacy of a candidate booster vaccine. Specifically, variant strain BCG-II, a minor component of BCG-Tokyo strain, which elicits weak immunity, was administered to guinea pigs. Vaccine-induced immunity and protection against Mycobacterium tuberculosis (Mtb) infection were evaluated using skin delayed-type hypersensitivity (DTH) and Mtb colony forming unit counts in organs, respectively. Candidate booster vaccine containing the mycobacterial DNA-binding protein 1 (MDP1) as antigen and CpG oligodeoxynucleotide G9.1 as adjuvant increased T-bet expression and IFN-γ production in human peripheral blood mononuclear cells. Intradermal administration of MDP1 or MDP1 and G9.1 to unimmunized guinea pigs produced DTH on MDP1-inoculated skin. Boosting BCG-II-primed guinea pigs with this protocol effectively enhanced DTH against MDP1 and protection against Mtb infection, particularly when combined with G9.1. The candidate vaccine may contribute to efforts to prevent tuberculosis.

MicroRNAs (miRNAs) are known to regulate gene expression and modulate cellular differentiation. MicroRNA-9/9* (miR-9/9*) and microRNA-124 (miR-124) are highly expressed in the central nervous system. In vivo function of miR-9/9* and miR-124 has been investigated in detail, whereas there remain some discrepancies regarding neural development. To this end, we electroporated miR-9/9*, miR-124 or miR-9/9*/124 expression plasmids into neonatal retinal progenitor cells (RPCs) in vivo and analyzed the fate of electroporated cells. Both miR-9/9* and miR-124 reduced the number of SOX9- and GS-positive cells and increased that of TUBB3-positive cells in the postnatal day 14 retina. No major effects on the proliferation and apoptosis of the electroporated cells were detected at least postnatal day 3. These indicated that miR-9/9* and miR-124 influence the cell fate of glial cells, thereby inducing their differentiation into neurons. Moreover, we found this cell fate modulation was occurred in RPCs indicating high-level expression of miRNA, but not in the low level. Our results strongly suggest that high-level miRNA overexpression is essential for directing cell fate by miR-9/9* and miR-124 interference.

Bacteria have been used for more than a century to treat solid tumors. Because solid tumors generate an anaerobic environment, we evaluated the anti-tumor effect of the obligate anaerobe strain KK378, derived from Lactobacillus casei (L. casei), using mice bearing head and neck cancer. Wild-type L. casei is a nonpathogenic bacterium that is commonly used in foods. Moreover, patients with head and neck squamous cell carcinoma often have multiple cancers and cervical lymph node metastasis that can be directly sensed beneath the skin. To establish the animal model bearing head and neck cancer, we inoculated each of human squamous cell carcinoma cell lines, SAS, HSQ89, and HSC2, on the back skin of BALB/cSlc-nu/nu mice. After tumor formation, L. casei KK378 was administered directly into the tumor, and tumor size and serum cytokine levels were analyzed. Mice injected with 108 cfu of L. casei KK378 showed reduction in tumor growth compared with PBS control; especially, the SAS tumor was significantly reduced (p = 0.008). Administered L. casei KK378 was detected in tumor tissues but not in normal tissues (liver, kidney, and lung) of SAS tumor-bearing mice, which was associated with increased blood cytokines (TNF-α, IFN-γ, IL-5, IL-10, and IL-12). Among these cytokines, the serum levels of IFN-γ and TNF-α were significantly increased (p < 0.05). In conclusion, L. casei KK378 infection may suppress tumor growth by inducing the host immune response. Direct injection of Lactobacillus into the tumor could be a potential strategy to treat head and neck squamous cell carcinoma.

Substantial variability exists among countries regarding the modes of death in pediatric intensive care units (PICUs). However, there is limited information on end-of-life care in Japanese PICUs. Thus, this study aimed to elucidate the characteristics of end-of-life care practice for children in a Japanese PICU.We examined life-sustaining treatment (LST) status at the time of death based on medical chart reviews from 2010 to 2014. All deaths were classified into 3 groups: limitation of LST (limitation group, death after withholding or withdrawal of LST or a do not attempt resuscitation order), no limitation of LST (no-limitation group, death following failed resuscitation attempts), or brain death (brain death group).Of the 62 patients who died, 44 (71%) had limitation of LST, 18 (29%) had no limitation of LST, and none had brain death. In the limitation group, the length of PICU stay was longer than that in the no-limitation group (13.5 vs 2.5 days; P = .01). The median time to death after the decision to limit LST was 2 days (interquartile range: 1-5.5 days), and 94% of the patients were on mechanical ventilation at the time of death in the limitation group.Although limiting LST was a common practice in end-of-life care in a Japanese PICU, a severe limitation of LST such as withdrawal from the ventilator was hardly practiced, and a considerable LST was still provided at the time of death.

Bacterial DNA comprising palindromic sequences and containing unmethylated CpG is recognized by toll-like receptor 9 of plasmacytoid dendritic cells (pDCs) and induces the production of interferon-α and chemokines, leading to the activation of a Th1 immune response. Therefore, synthetic equivalents of bacterial DNA (CpG oligodeoxynucleotides) have been developed for clinical applications. They are usually phosphorothioated for in vivo use; this approach also leads to adverse effects as reported in mouse models.Mucosal vaccines that induce both mucosal and systemic immunity received substantial attention in recent years. For their development, phosphodiester-linked oligodeoxynucleotides, including the sequence of a palindromic CpG DNA may be advantageous as adjuvants because their target pDCs are present right there, in the mucosa of the vaccination site. In addition, the probability of adverse effects is believed to be low. Here, we review the discovery of such CpG oligodeoxynucleotides and their possible use as mucosal adjuvants.

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150 kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a β-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N’-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.

Two wild-type substrains of a motile cyanobacterium Synechocystis sp. PCC 6803 show positive phototaxis toward a light source (PCC-P) and negative phototaxis away from light (PCC-N). In this study, we found that a novel two-component system of photoresponse is involved in the phototactic regulation. Inactivation of slr1212 (pixA), which encodes a photoreceptor histidine kinase, reverted the positive phototaxis of PCC-P to negative phototaxis, and inactivation of the downstream slr1213 (nixB) and slr1214 (nixC), which encode AraC-like transcription factor-type and PatA-type response regulators, respectively, reverted the negative phototaxis of PCC-N to positive phototaxis. Opposite effects of pixA and nixBC disruption implies an unexpected signal transduction pathway in the switching of positive and negative phototaxis. The blue/green-type cyanobacteriochrome GAF domain of PixA was expressed in Synechocystis and phycocyanobilin-producing Escherichia coli. The holoprotein covalently bound a chromophore phycoviolobilin and showed reversible photoconversion between the violet- (Pv, λ(peak) = 396 nm) and green-absorbing (Pg, λ(peak) = 533 nm) forms, although the protein from E. coli partially bound a precursor phycocyanobilin. These results were discussed with regard to an idea that PixA serves as a violet light receptor for switching of positive and negative phototaxis by transcriptional and functional regulation.

【目的】2 型糖尿病モデルマウス (KK-Ay マウス) を用い，霊芝菌糸体培養培地抽出物 (WER) の食後過血糖改善作用を検討した．さらに，α-グルコシダーゼ阻害薬との併用効果についても検証した． 【方法】KK-Ay マウスに WER を単独，またはボグリボース，アカルボースと同時に経口投与した後，糖負荷試験を行った． 【結果・考察】WER は，in vitro において α-グルコシダーゼ阻害作用を示した．また，KK-Ay マウスにおいてマルトース負荷後の血糖上昇を抑制した．正常血糖マウスにおいては，WER とボグリボースまたはアカルボースとの間に相互作用は認められなかったのに対して，KK-Ay マウスでは WER とボグリボースとの同時服用で血糖上昇抑制効果が消失した．しかし，投与間隔を 1 時間とすることでその効果は回復した．以上の結果より，WER は α-グルコシダーゼ阻害作用により食後過血糖改善作用を示すものの，WER と α-グルコシダーゼ阻害薬を同時服用した場合，薬物によってはその効果が減弱する可能性が示唆された．

The aim of the present study is to evaluate the creative attitude in reference to the expression of sand play. In this study, the word “creative attitude” doesn’t mean the ability in art, but means to having the experience where the maker express themselves and get their image refl ected in sand play, signifying the world around them. In the previous study, the author made two schemata to evaluate the creative attitude in reference to the expression of sand play. The first schema relates to the points of the questionnaire of “creative attitude,” named “a phase of the construction,” showing the richness of the visual impression. And the second schema has something to do with the points of the questionnaire of “openness,” named “a phase of the narrative,” showing the richness of the imagination. The structure of these schemata consists of six phases. After considering the result, the author presumes that 6 participants who used these schemata for the first time would understand them and these schemata would be available to them. But some problems were found in the schema. As the result of the analysis, the author extracts some factors which confused the participants. The author modifi es the previous schema and made two new ones. In this study, 5 participants evaluate 32 sand play works by using these new schemata. The result of the evaluation was compared with the score of the Rorschach Test for the purpose of verifying the validity of these schemata. As a result, some scores of Rorschach Test had something to do with the result of evaluation, but the author didn’t find sufficient results to consider the validity of the schema. After the analysis, the author extracts some factors of the problem in this experiment and a matter of the schema to be modifi ed. Accordingly, the author intends to improve these situations and examine the verifi cation of the validity of the schema in the next study.

【目的】霊芝菌糸体培養培地抽出物 (WER) は，マンネンタケ菌糸体をバガスおよび米糠を含む固形培地に接種し，子実体発生直前に培地と共に熱水抽出したものである．本研究では，WER の 2 型糖尿病態改善効果について検証した． 【方法】KK-Ay マウスに，WER を含む飼料を摂取させ，経時的に血糖値および血中インスリン量を測定した．また，インスリン感受性試験を行った．さらに，骨格筋および脂肪組織を摘出し GLUT4 蛋白質の発現量を測定した． 【結果・考察】WER 摂取群では，対照群と比較し血糖値および血中インスリン量が有意に低下した．また，WER 摂取群ではインスリンによる血糖値低下作用の増強が認められた．さらに，WER 摂取群の骨格筋および脂肪細胞膜では，GLUT4 蛋白質の発現増大が認められた．これらの結果から，WER は 2 型糖尿病マウスにおいて，インスリン抵抗性を改善し血糖上昇を抑制すること，またその機序の一部に GLUT4 の膜移行の促進が関与することが示唆された．

Background and purpose: The expression of multiple pharmacological phenotypes including α1L-adrenoceptor has recently been reported for α1-adrenoceptors. The purpose of the present study was to identify α1-adrenoceptor phenotypes in the irises of pigmented and albino rabbits. Experimental approach: Radioligand binding and functional bioassay experiments were performed in segments or strips of iris of pigmented and albino rabbits, and their pharmacological profiles were compared. Key results: [3H]-silodosin at subnanomolar concentrations bound to intact segments of iris of pigmented and albino rabbits at similar densities (approximately 240 fmol·mg−1 protein). The binding sites in the iris of a pigmented rabbit were composed of a single component showing extremely low affinities for prazosin, hydrochloride [N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053)] and 5-methylurapidil, while two components with high and low affinities for prazosin, RS-17053 and 5-methylurapidil were identified in irises from albino rabbits. In contrast, specific binding sites for [3H]-prazosin were not clearly detected because a high proportion of non-specific binding and/or low affinity for prazosin occurred. Contractile responses of iris dilator muscle to noradrenaline were antagonized by the above ligands, and their antagonist affinities were consistent with the binding estimates at low-affinity sites identified in both strains of rabbits. Conclusions and implications: A typical α1L phenotype with extremely low affinity for prazosin is exclusively expressed in the iris of pigmented rabbits, while two distinct phenotypes (α1A and α1L) with high and moderate affinities for prazosin are co-expressed in the iris of albino rabbits. This suggests that a significant difference in the expression of phenotypes of the α1-adrenoceptor occurs in the irises between the two strains of rabbits.

Alpha(1)-adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several alpha(1) antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) have been identified, among which the alpha(1A) and alpha(1D)-adrenoceptor subtypes have been regarded as the main targets of alpha(1) antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize alpha(1)-adrenoceptors pharmacologically, (i.e. all classical alpha(1)-adrenoceptor subtypes show high-affinity for the drug). However, we found that alpha(1)-adrenoceptors in the LUT show atypical low-affinity for prazosin. Therefore, the concept alpha(1L)-receptor, which indicates alpha(1)-adrenoceptor(s) showing low-affinity for prazosin has been introduced. A recent study demonstrated that the alpha(1L)-adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the alpha(1L)-adrenoceptor in the LUT is now re-defined as alpha(1A(L))-adrenoceptor. The physiological and pharmacological difference between classical alpha(1A(H),) and alpha(1A(L)) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological alpha(1A)-adrenoceptor blocking pharmacotherapy. Here, we briefly review the alpha(1)-adrenoceptors in the LUT with special reference to phenotype-based (pharmacome) analysis.

Distinct muscarinic acetylcholine receptor subtypes widely distribute in stomach tissues and are involved in many physiological functions. Although mRNA of M 1 subtype was found in gastric mucosa, the M 1 subtype has not been detected by conventional membrane binding assays. In the present study, muscarinic receptor subtypes in the rat stomach were reevaluated by using the tissue segment binding technique recently developed to recognize the inherent/native profiles of receptors without receptor environment perturbation. [ 3 H]-N-methylscopolamine (NMS) bound to muscarinic receptors in the intact segments of rat gastric mucosa and muscle layers. The muscarinic receptors in the mucosal segments were composed of M 1 , M 2 and M 3 subtypes, among which the M 1 subtype selectively showed high affinity for pirenzepine. However, in the membrane preparations, binding sites with high affinity for pirenzepine could not be detected. In the muscle layer, M 2 and M 3 subtypes, but not M 1 , were identified in tissue segment and conventional membrane binding assays. Western blotting analysis recognized the M 1 subtype in the membrane preparations of mucosal but not muscle layers. Chronic immobilization stress increased the M 3 subtype in mucosal and muscle layers and decreased the M 2 subtype in the muscle layer, whereas M 1 and M 2 subtypes in mucosal layer did not change after the stress. The current study shows that M 1 subtype occurs as a pirenzepine-high affinity entity in intact segments of rat gastric mucosa, but that it loses the affinity for pirenzepine upon homogenization. Careful identification of native in vivo muscarinic receptors may further elucidate their functions in stomach.

Background and purpose: The α1L-adrenoceptor has pharmacological properties that distinguish it from three classical α1-adrenoceptors (α1A, α1B and α1D). The purpose of this was to identify α1L-adrenoceptors in mice and to examine their relationship to classical α1-adrenoceptors. Experimental approach: Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and α1A-, α1B- and α1D-adrenoceptor gene knockout (AKO, BKO and DKO) mice. Key results: The radioligand [3H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of α1A-adrenoceptors and α1L-adrenoceptors. In membrane preparations of WT mouse cortex, however, [3H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of α1A-adrenoceptors alone. In contrast, [3H]-prazosin bound to two components having α1A-adrenoceptor and α1B-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked α1A-adrenoceptor profiles and BKO mice lacked α1B-adrenoceptor profiles. Noradrenaline produced contractions through α1L-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice. Conclusions and implications: α1L-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the α1L-adrenoceptor is one phenotype derived from the α1A-adrenoceptor gene. British Journal of Pharmacology (2008) 155, 1224–1234; doi:10.1038/bjp.2008.360; published online 22 September 2008

Triptycene derivatives bearing a pair of ionophores were synthesized and their rotational behaviors were observed with/without s -block metal cations as an external stimulus. In this study, oxyethylene-based ionophores were introduced at both bridgehead positions of triptycene as the stimulus receptors. The NMR experiments revealed the formation of complexes of the triptycenes with the s -block metal cations. We demonstrated that the rate (activation energy) of the internal rotation of the triptycyl moiety could be controlled by the formation of the complexes. We also found that the conformation of those complexes can be controllable with the species of metal cations.

Background and purpose: a1-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of a1-adrenoceptor phenotypes and to clarify their relationship. Experimental approach: Binding experiments with [ 3 H]-silodosin and [ 3 H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with a1-adrenoceptor-mediated prostate contraction. Key results: [ 3 H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [ 3 H]-prazosin at the same range of concentrations failed to bind to a1-adrenoceptors of intact segments. Binding sites of [ 3 H]-silodosin in intact segments were composed of a1L phenotype with low affinities for prazosin (pKi ¼ 7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-a,a-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and a1A-like phenotype with moderate affinity for prazosin (pKi ¼ 8.8) but high affinity for 5-methyurapidil and RS17053. In contrast, both radioligands bound to a single population of a1-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of ‘classical’ a1A-adrenoceptors (pKi for prazosin ¼ 9.8). The pharmacological profile of a1-adrenoceptor-mediated prostate contraction was in accord with the a1L phenotype observed by intact segment binding approach. Conclusions and implications: Three distinct phenotypes (a1L and a1A-like phenotypes in the intact segments and a classical a1A phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of a1A-adrenoceptors, but are not genetically different subtypes. British Journal of Pharmacology (2008) 155, 906–912; doi:10.1038/bjp.2008.318; published online 11 August 2008

The pharmacological profiles of alpha1-adrenoceptors for ketanserin, prazosin, silodosin, and BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride) were examined under different assay conditions. Among the tested antagonists and alpha1-adrenoceptors subtypes, ketanserin showed significantly lower affinity for the alpha1B-adrenoceptor subtype in intact tissue sampled from the rat tail artery, thoracic aorta, and cerebral cortex (functional pKB and binding pKi were approximately 6), than in cerebral cortex membrane preparations or whole cell and membrane preparations of alpha1B-adrenoceptor transfected human embryonic kidney 293T (HEK 293T) cells (pKi was approximately 8). In these tissues and cells, however, ketanserin showed a similar affinity (pKi = approximately 8) for alpha1A- and alpha1D-adrenoceptors even though the assays were conducted under different conditions. In contrast, the affinities of alpha1A-, alpha1B-, and alpha1D-adrenoceptors for prazosin, silodosin, and BMY 7378 did not significantly change under different assay conditions and in different tissues. The present study reveals that the pharmacological profiles of native alpha 1B-adrenoceptors for ketanserin is strongly influenced by the assay conditions and suggest that antagonist affinity is not necessarily constant.

Background and purpose: In addition to α1A, α1B and α1D-adrenoceptors (ARs), putative α1L-ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the α1A-AR and clarify its pharmacological profile using a radioligand binding assay.

糖尿病は体内の酸化ストレスを増大させ，一過性脳虚血による脳障害を増悪させることが報告されている。本研究では，ストレプトゾトシン糖尿病態（DM）ラットを用い，一過性脳虚血処置による脳障害に対する霊芝菌糸体培養培地抽出物（WER）の効果を検討した。正常血糖（non-DM）およびDMラットに，WER（1 g/kg）または蒸留水を1日1回2週間経口投与した後，体内の酸化ストレス度，脳組織中の過酸化脂質含量および抗酸化酵素活性を測定した。また，ラットに中大脳動脈閉塞/再潅流（MCAO/Re）処置を行い，神経症状の評価および脳梗塞巣体積の測定を行った。その結果，DMラットでは体内の酸化ストレス度の上昇，脳組織中の過酸化脂質含量の増加および抗酸化酵素活性の低下がみられた。WERを投与したDMラットでは，これらの値が正常レベルに維持されていた。さらに，DMラットでは，non-DM群と比較し，MCAO/Re後の神経症状の悪化および脳梗塞巣体積の顕著な増大が認められたが，WERの投与により，脳障害の増悪がほぼ完全に抑制された。以上の結果から，WERは糖尿病における酸化ストレス状態を改善し，一過性脳虚血に対して強い脳保護効果を示すことが明らかになった。

脳虚血では，局所における過剰の活性酸素種の産生によりアポトーシスが誘発される．本研究では，酸化ストレスによる神経障害に対する霊芝菌糸体培養培地抽出物 (WER) の保護効果について，培養細胞およびマウス脳虚血モデルを用いて検討した． PC12 細胞に H2O2 処理を行い，誘発される細胞死に対する WER の作用を MTT assay および TUNEL 法により評価した．また，マウスに WER (1 g/kg) を単回または 7 日間経口投与した後，低酸素脳虚血処置を行い，神経症状，脳梗塞巣体積およびアポトーシスの評価を行った． PC12 細胞を用いた実験において，WER は H2O2 処理後の細胞生存率を上昇させ，アポトーシス陽性細胞数を減少させた．WER を 7 日間経口投与したマウス群では，対照群と比較し，虚血後の神経症状の改善が認められ，また脳梗塞巣体積およびアポトーシス陽性細胞数が有意に減少した．これらの結果から，WER は酸化ストレスによるアポトーシスを抑制し，脳虚血障害に対して保護作用を示すことが示唆された．

糖尿病では，持続的な高血糖状態による酸化ストレスが病状の進行や合併症の誘発に密接に関与する．そこで，霊芝菌糸体培養培地抽出物 (WER) の抗酸化活性を調査し，ストレプトゾトシン (STZ) 誘発糖尿病マウスを用い，WER の糖尿病態改善効果について検証した．WER の O2? 消去能および過酸化脂質産生抑制能を調査した．また，STZ 糖尿病マウスに WER (1 g/kg) を約 2 ヶ月間 1 日 1 回経口投与し，血糖値，体内酸化ストレス度，臓器の過酸化脂質含量および抗酸化酵素 (catalase, SOD, GPx) の活性について検討した．WER は濃度依存的な O2? 消去能および過酸化脂質産生抑制能を示した．WER を投与した STZ 糖尿病マウスでは対照群に比べ血糖値および血中酸化ストレス度の有意な低下が認められた．また，WER は腎臓および肝臓の過酸化脂質含量および抗酸化酵素活性を正常群と同レベルに維持し，組織学的障害を軽減した．これらの結果から，WER は糖尿病における高血糖および酸化ストレスを低下させ，臓器障害を予防・改善する可能性が示された．, 原著, 本文データは学会の許諾に基づきJ-STAGEより複製したものである。

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.

Background Hyperoxia is a known cause of diffuse alveolar damage (DAD). We previously reported the transcript profiling of DAD induced by hyperoxia exposure in mouse lungs and showed that the gene expression of myelocytomatosis oncogene ( c-Myc ) was significantly upregulated whereas that of surfactant-associated protein ( SP ) -C was downregulated. However, the mechanism underlying hyperoxia-induced DAD is not well understood. Methods The hyperoxia-induced changes in SP-A / B / C / D , c-Myc , B-cell chronic lymphocytic leukemia/lymphoma ( Bcl ) - 2, and Bcl- 2 -associated X protein ( Bax ) expression in mouse lungs were examined by cDNA microarray analysis. The expression levels of the above mentioned genes, cell viability, caspase activity, and reactive oxygen species (ROS) production were also examined in the human lung adenocarcinoma cell line A549 and mouse fibroblast-like cell line NIH/3T3. Results Hyperoxia induced a decrease in SP-C/A expression in mouse lungs, and SP-C downregulation was also confirmed in A549 cells. In addition to enhanced c-Myc expression, Bax expression also increased following exposure of the mice to hyperoxia. In vitro analysis showed that expression of these genes is regulated in a cell-type-dependent manner, i.e ., upregulation of c-Myc in NIH/3T3 cells and Bax in A549 cells occurred regardless of whether there was a similar decrease in cell viability and increase in caspase-3/7 activation in response to hyperoxia. ROS production and caspase-8 activation were also observed in both cells. Conclusions We concluded that hyperoxia induces ROS production and cell death in lung tissues through a cell-type specific mechanism involving the upregulation of c-Myc / Bax, and caspase-8 and -3/7 activation-dependent pathways, thereby leading to the development of DAD.

Alpha(1)-adrenoceptors are widely distributed in the human body and play important physiologic roles. Three alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) have been cloned and show different pharmacologic profiles. In addition, a putative alpha(1)-adrenoceptor (alpha(1L) subtype) has also been proposed. Recently, three drugs (tamsulosin, naftopidil, and silodosin) have been developed in Japan for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. In this review, we describe recent alpha(1)-adrenoceptor subclassifications and the pharmacologic characteristics (subtype selectivity and clinical relevance) of alpha(1)-adrenoceptor antagonists.

The radioligand binding assay technique is an extremely powerful tool for studying receptors. It allows an analysis of the interactions of hormones, neurotransmitters, and related drugs with their receptors. Most of the binding assays have widely been applied to crude membrane fractions prepared from many tissues, but in the conventional method, there are some limitations such as a yield loss of receptor-bearing membranes and a change in receptor environment upon homogenization and fractionation. Recently, in order to overcome these problems, a binding assay has been developed using intact tissue segments. This article presents a brief overview of the tissue segment binding assay that has been developed mainly in our department. Practical guidelines for setting up this new assay are presented, including segment preparation, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. The unique advantages and disadvantages of the tissue segment binding method are discussed in comparison with those of conventional membrane binding methods. We suggest that the tissue segment binding method is a powerful tool for detecting the native properties of receptors occurring in tissues and cells without altering their environment. Keywords:: tissue segment binding method, receptor, pharmacome analysis, pharmacomics, radioligand

We examined reserpine-induced chemical denervation supersensitivity with special reference to alpha-1 adrenoceptor (AR) subtypes. Chronic treatment with reserpine for 2 weeks depleted noradrenaline in the tail artery and spleen of rats. Noradrenaline in the thoracic aorta was negligible before and after reserpine treatment. The treatment with reserpine produced supersensitivity in the contractile responses of the rat tail artery to phenylephrine, 5-HT and KCl, resulting in leftward shift of concentration–response curves (11.6-, 2.5- and 1.1-fold at EC50 value, respectively). These results suggest a predominant sensitization of the alpha-1 AR-mediated response by reserpine treatment. BMY 7378 at a concentration (30 nM) specific for blocking the alpha-1D AR subtype, but not KMD-3213 at a concentration (10 nM) selective for blocking the alpha-1A AR subtype, inhibited the supersensitivity of the phenylephrine-induced response in the reserpine-treated artery. On the other hand, the response to phenylephrine in reserpine-untreated artery was selectively inhibited by the same concentration of KMD-3213, but not by BMY 7378. Prazosin, a subtype-nonselective antagonist, blocked the responses to phenylephrine with the same potency, regardless of reserpine treatment. In the thoracic aorta and spleen, no supersensitivity was produced in the responses to phenylephrine by reserpine treatment. In a tissue segment-binding study using [3H]-prazosin, the total density and affinity of alpha-1 ARs in the rat tail artery were not changed by treatment with reserpine. However, alpha-1D AR with high affinity for BMY 7378 was significantly detected in reserpine-treated tail artery, in contrast to untreated artery. Decreases in alpha-1A AR with high affinity for KMD-3213 and alpha-1B AR with low affinities for KMD-3213 and BMY 7378 were also estimated in reserpine-treated tail artery. Alpha-1D AR mRNA in rat tail artery increased to three-folds by reserpine treatment, whereas the levels of alpha-1A and 1B mRNAs were not significantly changed. The present results suggest that chronic treatment with reserpine affects the expression of alpha-1 AR subtypes of rat tail artery and that the induction of alpha-1D ARs with high affinity for catecholamines is in part associated with reserpine-induced supersensitivity. Keywords: Supersensitivity, reserpine, chemical denervation, alpha-1 adrenoceptor (AR), intact tissue segment binding, mRNA, rat tail artery Introduction Supersensitivity is one of the classical and pharmacological phenomena and is defined as an increase in functional affinity (that is, a decreased EC50) for agonists (Fleming et al., 1973; Fleming, 1976; Westfall, 1981). Supersensitivity may be caused by a variety of procedures, including surgical and chemical denervation and chronic treatment with antagonists (Westfall, 1981; Insel, 1989). The one common feature of these treatments has been reported that they all cause chronic interruption of neuronal stimulation on target cells. Reserpine depletes catecholamine in the adrenergic nerve due to inhibition of catecholamine reuptake by binding the pre-junctional storage vesicle for catecholamine (Berkowitz et al., 1971; Stitzel, 1976; Giachetti & Shore, 1978). Chronic treatment with reserpine is known to cause supersensitivity of the adrenergic and nonadrenergic responses in sympathetically innervated tissues, which has been respectively called specific and nonspecific supersensitivities (Fleming et al., 1973). In extensive studies, several mechanisms underlying the supersensitivity have been proposed; increases in the density or affinity of postjunctional receptors (Bito & Dawson, 1970; Colucci et al., 1981), changes in the resting membrane potential (Fleming & Westfall, 1975; Abel et al., 1981; Fleming, 1987) and alterations of calcium homeostasis (Hudgins & Harris, 1970; Carrier, 1975). Recently, alpha-1 ARs have been classified into at least three subtypes, alpha-1A, -1B and -1D, in molecular and pharmacological evaluations (Minneman et al., 1994; Hieble et al., 1995; Michel et al., 1995; Piascik & Perez 2001). These subtypes distribute distinctly and play key roles in adrenergic functions in many tissues (McGrath & Wilson, 1988; Muramatsu et al., 1995; 1998; Graham et al., 1996; Daly et al., 2002). In rats, for example, the adrenergic contraction of the tail artery is predominantly mediated by the alpha-1A AR subtype (Lachnit et al., 1997; Murata et al., 1999; Gisbert et al., 2003), while the adrenergic contractions of the thoracic aorta and spleen are mainly through alpha-1D and alpha-1B AR subtypes, respectively (Burt et al., 1995; Muramatsu et al., 1995; Buckner et al., 1996; Saussy et al., 1996). Recent studies further developed various drugs specific for each subtype. Prazosin is a classical antagonist which shows no subtype selectivity (Muramatsu et al., 1995; Hancock, 1996). BMY 7378 and KMD-3213 are well characterized as highly selective antagonists for alpha-1D and alpha-1A AR subtypes, respectively (Goetz et al., 1995; Shibata et al., 1995; Saussy et al, 1996; Yamagishi et al., 1996; Murata et al., 1999). Furthermore, several agonists including phenylephrine show relatively high affinity for the alpha-1D subtype (Lomasney et al., 1991; Buckner et al., 1996; Graham et al., 1996; Piascik & Perez, 2001). Stassen et al. (1998) evaluated the relationship between the presence of adrenergic nerves and the presence of alpha-1 ARs in the arterial trees of rat, and suggested that the presence of alpha-1 AR subtypes may be closely related to the sympathetic innervation. They also demonstrated that chemical sympathectomy of rats with 6-hydroxydopamine caused a distinct reduction in alpha-1A AR subtype, concomitant with a marked reduction of noradrenaline content. We hypothesized that the supersensitivity may in part reflect the changes in expression of alpha-1 AR subtypes. In the present study, we examined a possible relationship between reserpine-induced supersensitivity and alpha-1 AR subtypes. To this end, we analyzed the concentration–contractile response curve for phenylephrine, the binding of [3H]-prazosin, the effects of the alpha-1 AR subtype-selective ligands KMD-3213 and BMY 7378, and the mRNA levels of alpha-1 AR subtypes.

The binding kinetics of [3H]-prazosin were measured using intact segments of rat tail artery (RTA) and thoracic aorta (RAO), and the data were compared with those obtained using a conventional membrane ligand-binding method. In intact RTA and RAO segments, [3H]-prazosin bound reversibly in a time-dependent and receptor-specific manner at 4 degrees C to alpha-1 adrenoceptors (ARs) of the plasma membrane, with affinities (pKD): 9.5 in RTA; 9.9 in RAO) that were in agreement with values estimated by a conventional membrane ligand-binding method. However, nonspecific binding was considerably higher in RAO than RTA, failing to detect clearly the specific binding at high concentrations (>300 pm) of [3H]-prazosin in binding experiments with RAO segments and membranes. The abundance of receptor in the RTA and RAO (Bmax mg-1) of total tissue protein), estimated using the tissue segment-binding approach (527+/-14 fmol mg-1 for RTA; 138+/-4 fmol mg-1 for RAO), was about 25-fold higher than values estimated using a conventional membrane-binding method (22+/-5 fmol mg-1) for RTA; 5+/-1 fmol mg-1 for RAO). Binding competition experiments using intact tissue segments or membranes derived from RTA tissue yielded comparable data, indicating a coexistence of alpha-1A AR (high affinity for prazosin, KMD-3213 and WB4101 and low affinity for BMY 7378) and alpha-1B AR (high affinity for prazosin but low affinity for KMD-3213, WB4101 and BMY 7378). In RAO tissue, careful evaluation of the tissue segment-binding assay revealed the coexpression of alpha-1B AR (high affinity for prazosin, but low affinity for KMD-3213 and BMY 7378) and alpha-1D AR (high affinity for prazosin and BMY 7378, but low affinity for KMD-3213), whereas the membrane-binding approach failed to detect these receptor subtypes with certainty. The present study indicates that previous estimates of alpha-1 AR density and alpha-1 AR subtypes obtained by a conventional membrane-binding approach, as opposed to our improved tissue segment-binding assay, may have substantially underestimated the abundance of receptors present in arterial tissues, and may have failed to identify accurately the presence of receptor subtypes. Advantages and disadvantages of the tissue segment-binding approach are discussed.British Journal of Pharmacology (2004) 141, 468-476. doi:10.1038/sj.bjp.0705627

We established three human embryonic kidney (HEK) 293 cell lines stably expressing alpha(1)-adrenoceptor (AR) subtypes, one (alpha(1A), (1B)-AR) coexpressing both receptors and the other two (alpha(1A)-AR and alpha(1B)-AR) expressing each receptor in isolation. In the alpha(1A), (1B)-AR cells, both receptors were clearly distinguished by the alpha(1A)-selective ligands (-)-1(3-hydroxypropyl)-5-((2R)-2-([2-(2,2,2-trifluoroethyl]oxy]phenyl)oxy)ethyl]amino)propyl)-2,3-dihydro-1H-indole-7-carboxamide (KMD-3213) and methoxamine, but not by the subtype-nonselective ligands prazosin and phenylephrine. In all three cell lines, phenylephrine caused a concentration-dependent increase in inositol phosphates and an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activation. However, there was a 2-fold or greater maximal response to phenylephrine and a somewhat higher agonist potency in ERK1/2 activation in the alpha(1A,1B)-AR cells, compared with the responses of cells expressing either receptor individually (alpha(1A)-AR or alpha(1B)-AR). Furthermore, the antagonistic affinities of prazosin (pK(b) of 10.1) and KMD-3213 (9.4) for inhibiting the phenylephrine response were intermediate between the values for inhibition in alpha(1A)-AR cells (prazosin, 9.3; KMD-3213, 10.5) and alpha(1B)-AR cells (prazosin, 11.0; KMD-3213, 8.1). The inhibitor pK(b) values in alpha(1A), (1B)-AR also differed from their ligand binding affinities measured in alpha(1A)-AR and alpha(1B)-AR cells. In contrast, the alpha(1A)-selective agonist methoxamine, which did not activate alpha(1B)-AR cells, stimulated either alpha(1A,) (1B)-AR or alpha(1A)-AR cells with a comparable potency and maximum effectiveness. Our data indicate that when coexpressed in the same cell, the activation of common pathways by individual AR receptor subtypes by a nonselective agonist can exhibit enhanced responsiveness and a distinct antagonist affinity compared with the parameters for the same receptors, when expressed alone in the same cell background.

We investigated subtypes of alpha-1 adrenoceptor (AR) in rabbit ocular tissues using reverse transcription-polymerase chain reaction (RT – PCR), in situ hybridization (ISH) and binding studies. Competitive RT – PCR assays specific for the subtypes of alpha-1 AR revealed that the mRNA expression of alpha-1a AR was dominant, and that of each alpha-1b and alpha-1d was less than 10% and 0.5% of total alpha-1 ARs mRNA, respectively, in the iris, ciliary body, choroid and retina. In alpha-1a AR splice isoform-specific RT – PCR assays, we found a distinct proportion of each isoform mRNA in the iris, ciliary body and choroid. The results of the ISH assays for alpha-1a AR subtype showed that hybridization signals were clearly observed in the iris dilator muscle and in the epithelium of the ciliary processes. In binding studies, alpha-1A AR was a dominant subtype in the iris, choroid and retina in contrast to the ciliary body that had more alpha-1B than alpha-1A AR subtype at protein level. Keywords: Alpha-1 adrenoceptor, eye, iris, dilator muscle, ciliary body Introduction Alpha-1 ARs constitute a heterogeneous family of receptors and play critical roles in the regulation of the sympathetic system. Molecular cloning techniques revealed the existence of three alpha-1 AR subtypes (alpha-1a, alpha-1b and alpha-1d AR) in many species (Cotecchia et al., 1988; Schwinn et al., 1990; Lomansney et al., 1991; Perez et al., 1991) and pharmacological studies suggested that these recombinant subtypes correspond to native alpha-1A, alpha-1B, and alpha-1D AR subtypes, respectively (Bylund et al., 1994; Hieble et al., 1995). Furthermore, splice isoforms of the alpha-1a AR subtype were found in humans; alpha-1a HSA.1-, 2-, 3- and 4-AR (Hirasawa et al., 1995; Chang et al., 1998) and in rabbits; alpha-1a OCU.1-, 2- and 3-AR (Suzuki et al., 2000). Alpha-1 ARs play important functions in the eyes. The contraction of the iris dilator muscle in rabbit eyes is mediated by alpha-1 ARs (Konno & Takayanagi, 1986; Nakamura et al., 1999). In vivo studies have revealed that alpha-1 ARs are involved in the regulation of intraocular pressure (IOP) in rabbits and monkeys (Kiuchi et al., 1992; Nishimura et al., 1993; Wang et al., 1997; Zhan et al., 1998; Okada & Gregory, 2001). Moreover, alpha-1 ARs have been found to regulate cellular functions in cultured endothelial cells from various ocular regions in several species (Walkenbach et al., 1992; Moroi-Fetters et al., 1995; Ryan et al., 1998). However, which subtype(s) of alpha-1 AR is involved in the ocular functions remains to be clarified. The purpose of this study was to identify and characterize alpha-1 AR subtypes in rabbit ocular tissues by RT – PCR, ISH and binding studies.

The release of calcium ions (Ca(2+)) from their intracellular stores is essential for the fertilization of oocytes of various species. The calcium pools can be induced to release Ca(2+) via two main types of calcium channel receptor: the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor. Starfish oocytes have often been used to study intracellular calcium mobilization during oocyte maturation and fertilization, but how the intracellular calcium channels contribute to intracellular calcium mobilization has never been understood fully, because these molecules have not been identified and no specific inhibitors of these channels have ever been found. In this study, we utilized a novel IP(3)R antagonist, the "IP(3) sponge," to investigate the role of IP(3) during fertilization of the starfish oocyte. The IP(3) sponge strongly and specifically competed with endogenous IP(3)R for binding to IP(3). By injecting IP(3) sponge into starfish oocyte, the increase in intracellular calcium and formation of the fertilization envelope were both dramatically blocked, although oocyte maturation was not blocked. To investigate the role of IP(3)R in the starfish oocyte more precisely, we cloned IP(3)R from the ovary of starfish, and the predicted amino acid sequence indicated that the starfish IP(3)R has 58-68% identity to mammalian IP(3)R types 1, 2, and 3. We then raised antibodies that recognize starfish IP(3)R, and use of the antibodies to perform immunoblot analysis revealed that the level of expression of IP(3)R remained unchanged throughout oocyte maturation. An immunocytochemical study, however, revealed that the distribution of starfish IP(3)R changes during oocyte maturation.

Serotonin (5-HT2) antagonists show high affinity for the α1-adrenoceptor (α1-AR) in addition to the 5-HT2 receptor. In the present study we compared the pharmacological characteristics of a new 5-HT2 antagonist sarpogrelate and its active metabolite M-1 with those of ketanserin on human recombinant α1-AR subtypes. In the binding study, sarpogrelate, M-1 and ketanserin produced concentration-dependent inhibition of 3H-prazosin binding to α1-ARs. Among the three drugs, ketanserin showed the highest affinity for α1a-, α1b- and α1d-ARs (pKi 8.0, 8.3 and 7.6, respectively). Sarpogrelate had a relatively low affinity for the three subtypes (6.3 , 6.4 and 6.3, respectively) and M-1 showed medium affinity (7.1, 7.1 and 6.1, respectively). Chinese hamster ovary (CHO) cells expressing each α1-AR subtype showed concentration-dependent inositol phosphate (IP) accumulation in response to phenylephrine. The concentration response curves were shifted to the right by three drugs, and the pKb values were close to the pKi values in the binding study. In addition to these effects, sarpogrelate and M-1, but not ketanserin produced an increase in the basal IP level of α1d-expressed CHO cells, although the increase was less than that of phenylephrine. The present results indicate that sarpogrelate and M-1 have antagonistic activity to the three α1-AR subtypes, but their affinities are significantly lower than those of ketanserin.

The polysaccharide chitin is effectively hydrolyzed and utilized as a carbon and nitrogen source by the Gram-positive bacterium Paenibacillus sp. strain FPU-7. ChiW is a unique cell-surface-expressed chitinase among the Paenibacillus sp. strain FPU-7-secreted chitinases. An N-terminally truncated ChiW protein, primarily comprised of the two catalytic domains of the full-length protein, was successfully overexpressed in Escherichia coli, purified as a functional recombinant protein with a molecular mass of approximately 98 kDa and crystallized. Preliminary X-ray analysis showed that the crystal diffracted to 1.93 A resolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 112.1, b = 128.2, c = 162.6 A, suggesting the presence of two molecules in an asymmetric unit.

Background CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated TH1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. Methods TH1 and TH2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination. Results G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of TH1, but not TH2, -type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. Conclusions G9.1 is a promising mucosal adjuvant for induction of pDC-mediated TH1 immunity.

Using a microphysiometer with synchronized valve switching, we investigated real-time acid extrusion from Chinese hamster ovary (CHO) cells in which human α1 adrenoceptor (AR) is stably expressed, in response to noradrenaline (NA). In the cells expressing α1a AR, the time course of extracellular acidification after stimulation had two phases; in the first phase it transiently reached a rate several times greater than the base rate with a peak at around 10 s, and in the second it increased to 2 times the base rate and reached a plateau in 2 min. Both phases showed a concentration-dependent increase of acidification rate in response to NA, but had distinct pEC50 values; 5.6 for the transient phase and 7.2 for the steady phase. In the cells expressing α1b AR, the transient phase was not detected but the steady phase was observed. The pEC50 value was 7.1, although the magnitude of the response was much smaller than that with α1a AR. Both 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and HOE642 inhibited the acid extrusion response by either AR in a concentration-dependent manner. EIPA and HOE642 had high pIC50 values (7.4 and 7.3, respectively) for inhibition of the transient phase response via α1a AR. In the inhibition of the steady phase response via α1a AR, both drugs revealed the presence of two components in the response; one had high pIC50 values (8.1 and 8.2 for EIPA and HOE642, respectively) and the other had low pIC50 values (5.6 and 6.0, respectively). In contrast, the steady phase response via α1b AR was inhibited by EIPA and HOE642 with low pIC50 values (5.3 and 5.9, respectively). As Ca2+ was depleted, the α1a AR-induced transient phase disappeared, while the steady phase was not affected. These results suggest that α1a AR drives two acid extrusion systems in CHO cells upon stimulation; one elicits the transient response, which is largely mediated by an EIPA/HOE642-sensitive and Ca2+-dependent Na+-H+ exchanger (NHE), presumably NHE1, and the other induces the steady acid extrusion that is mediated by NHE1 and another NHE which has low sensitivity to both EIPA and HOE642. α1b AR drives only the steady phase acid extrusion response, which is mainly mediated by NHEs other than NHE1. Until recently, extracellular pH was believed to be maintained within a narrow range of around 7.4 by homeostatic mechanisms. In fact, deviation of the pH from this value in body fluid (either acidosis or alkalosis) leads to serious malfunction of cellular processes (Adrogue & Madias, 1998). However, investigations in the past few years have revealed that extra- as well as intracellular pH are not so constant as generally thought, and it has even been hypothesized that H+ acts, not secondarily to metabolic changes or ionic membrane transport, but as a signalling molecule to initiate or relay intercellular communications (Chesler, 1990; Deitmer & Rose, 1996). In general, the buffering capacity of the extracellular space and net movement of acid/base equivalents determine extracellular pH. In the former, the intrinsic buffer value and volume of extracellular fluid are major factors. In the latter, many molecules such as channels, pumps and transporters are involved (Chesler, 1990; Deitmer & Rose, 1996). Although several methods such as pH-sensitive microelectrodes and magnetic resonance are generally used to estimate extracellular pH, these methods have certain drawbacks and difficulties, in particular their poor quantitative evaluation and/or low time resolution (Chesler, 1990; Smith et al. 1998). The microphysiometer is a potentiometric system that is able to detect small changes of pH in extracellular fluid accurately (McConnell et al. 1992). It has been employed to monitor cellular responses to various stimuli quantitatively; however, it does not produce real-time recordings (Taniguchi et al. 1999). We employed a microphysiometer with synchronized valve switching to estimate the real-time change of extracellular pH in Chinese hamster ovary (CHO) cells expressing human α1 adrenoceptor (AR). We report a rapid acid extrusion from CHO cells in response to noradrenaline (NA), which is mediated by a Na+-H+ exchanger (NHE). We characterized the acid extrusion response and found that it occurs in two phases, involving at least two NHEs.

形態: カラー図版あり, Physical characteristics: Original contains color illustrations, Funding: This study was partially supported by the "High-Quality Protein Crystal Growth Experiment on JEM" promoted by JAXA (Japan Aerospace Exploration Agency)., Accepted: 2016-11-13, 資料番号: PA1710029000

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene ( chiW ) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes ( chiA to chiF and chiW ) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

We have studied the antagonist action of prazosin and KMD-3213 in a constitutively active mutant of the human alpha-1a adrenoceptor in which Ala(271) was substituted to Thr and was expressed in CHO cells. Inverse agonism was characterized by up-regulation of receptor density, a decrease in basal GTPgammaS binding, and a reduction in basal inositol-1,4,5-trisphosphate (IP(3)) level. According to the above criteria, prazosin acted as an inverse agonist, whilst KMD-3213 behaved as a neutral antagonist. Compared with the wild-type receptor, mutant receptor exhibited single affinity sites for [(3)H]-prazosin, [(3)H]-KMD and the non-radioactive ligands tested, and displayed significantly higher affinities for several agonists but not for the two antagonists. Administration of KMD-3213 to prazosin-treated CHO cells expressing the mutant receptor reversed the inverse agonism of prazosin resulting in rapid increases in cellular IP(3), in intracellular [Ca(2+)] and in the rate of extracellular acidification. These results indicated that a neutral antagonist can reverse the action of an inverse agonist at the receptor site. The distinct properties of inverse agonist and neutral antagonist in affecting receptor function may be important for the clinical use of such antagonists.