Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ejpb.2022.10.020

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s11095-022-03337-4

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Other Link： https://link.springer.com/article/10.1007/s11095-022-03337-4/fulltext.html

Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s11095-022-03325-8

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Other Link： https://link.springer.com/article/10.1007/s11095-022-03325-8/fulltext.html

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ejpb.2022.02.002

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.xphs.2021.11.020

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ejpb.2021.08.013

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Language：English   Publishing type：Research paper (scientific journal)  

The improvement effect of the combined use of spermine (SPM), a polyamine, with sodium taurocholate (STC) on the pulmonary drug absorption was investigated utilizing poorly absorbable drugs with various molecular sizes in rats. The pulmonary absorption of rebamipide, a low molecular but poorly absorbable drug after oral administration, was significantly improved by the combined use of SPM with STC (SPM-STC formulation), while poly- L-lysine did not show a significant change in rebamipide absorption from the lungs. Furthermore, the safety of the SPM-STC formulation for the lungs was assessed in rats by the histopathological study and any local toxicity was not observed while poly-L-lysine, a typical chemical causing the toxicity for the epithelial cells, provided several histopathological changes. In addition, the SPM-STC formulation significantly improved the pulmonary absorption of fluorescein isothiocyanate dextran 4 (FD-4, Mw ca 4000) and interferon-α (IFN-α, Mw ca 25,000) as well. Our present results clearly indicated that the SPM-STC formulation significantly improved the pulmonary absorption of poorly absorbable small and large molecular drugs without any harmful effects on the lungs. Therefore, the SPM-STC formulation would be a useful one for the pulmonary absorption of drugs, specifically macromolecular ones, which are very difficult to be absorbed after oral administration.

DOI： 10.1016/j.xphs.2021.06.015

PubMed

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.xphs.2021.09.014

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Language：English   Publishing type：Research paper (scientific journal)  

Glioblastoma multiforme (GBM) is the most prevalent malignant primary brain tumor with a high recurrence rate. Despite multimodal therapy including surgical resection, chemotherapy, and radiotherapy, the median survival time after the initial diagnosis of GBM is approximately 14 months. Since cancer stem cells (CSCs) are considered the leading cause of cancer recurrence, glioblastoma stem cell-targeted therapy is a promising strategy for the treatment of GBM. However, because CSC heterogeneity has been implicated in the difficulties of CSC-target therapy, more in-depth knowledge of CSC biology is still required to develop novel therapies. In this study, we established single cell-derived tumorspheres from human glioblastoma U87MG cells. One of these tumorspheres, P4E8 clone, showed CSC-like phenotypes, such as self-renewal capacity, expression of CSC markers, resistance to anti-cancer agents, and in vivo tumorigenicity. Therefore, we used P4E8 cells as a cell-based model of glioblastoma stem cells (GSCs). Gene expression analysis using microarray indicated that the most highly expressed genes in P4E8 cells compared to the parental U87MG were PC3-secreted microprotein (MSMP). Furthermore, MSMP was expressed in patient-derived GSCs and human glioma tissues at the protein level, implying that MSMP might contribute to glioma development and progression.

DOI： 10.1248/bpb.b20-00868

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

Among drugs in development and/or in market, there are poorly water-soluble and poorly lipid-soluble compounds. Rebamipide, classified into BCS class IV, is one of those drugs which provide very low bioavailability and/or the difficulty of formulation for oral administration. Because of its low solubility in available lipoidal excipients, it was impossible to prepare an adequate SNEDDS formulation of rebamipide. Then, we tried to increase the solubility of rebamipide in lipoidal excipients for preparing a more practical SNEDDS formulation by making the complex with its counter ion, tetrabutylphosphonium hydroxide (TBPOH) or NaOH. Rebamipide concentration in ethanol was proportionally increased with the increment of TBPOH or NaOH added, indicating that the formation of complex with a counter ion should contribute to the solubilization of rebamipide in ethanol. Both Rebamipide-TBPOH complex (Reb-TBPOH) and Rebamipide-NaOH complex (Reb-NaOH) obtained by lyophilization showed no endothermic peak in DSC and no diffraction peak in XRPD, suggesting that the solid state of both complexes should be amorphous. Reb-TBPOH maintained the dissolution of rebamipide in SNEDDS vehicle (Capryol 90:Cremophor EL:Transcutol P = 4:3:3) at 20 mg/g at least for 28 days, while Reb-NaOH did it at 10 mg/g. In vitro dissolution study showed that Reb-TBPOH SNEDDS and Reb-NaOH SNEDDS containing rebamipide at 10 mg/g maintained the complete dissolution of rebamipide in FaSSIF (intestinal luminal condition). In the gastric luminal condition (pH3.9 acetate buffer), the high concentration, close to the complete dissolution, was transiently observed and quickly decreased to one-sixth of the maximum, but it was still around 70 times higher than that of the crystalline powder. The additional utilization of Eudragit EPO for SNEDDS preparations of both complexes successfully maintained the high concentrations of rebamipide in the gastric luminal condition. In vivo oral absorption studies clearly indicated that SNEDDS preparations utilizing Reb-counter ion complex successfully improved rebamipide absorption.

DOI： 10.1016/j.ejps.2021.105721

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

Elucidating fine architectures and functions of cellular and synaptic connections requires development of new flexible methods. Here, we created a concept called the "battle of transgenes," based on which we generated strategies using genetically engineered battles of multiple recombinases. The strategies enabled split-tunable allocation of multiple transgenes. We demonstrated the versatility of these strategies and technologies in inducing strong and multi-sparse allocations of multiple transgenes. Furthermore, the combination of our transgenic strategy and expansion microscopy enabled three-dimensional high-resolution imaging of whole synaptic structures in the hippocampus with simultaneous visualizations of endogenous synaptic proteins. These strategies and technologies based on the battle of genes may accelerate the analysis of whole synaptic and cellular connections in diverse life science fields.

DOI： 10.1016/j.isci.2020.101248

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Targeting immune checkpoint proteins has recently gained substantial attention due to the dramatic success of this strategy in clinical trials for some cancers. Inducible T-cell co-stimulator ligand (ICOSLG) is a member of the B7 family of immune regulatory ligands, expression of which in cancer is implicated in disease progression due to regulation of antitumor adaptive immunity. Although aberrant ICOSLG expression has been reported in glioma cells, the underlying mechanisms that promote glioblastoma (GBM) progression remain elusive. METHODS: Here, we investigated a causal role for ICOSLG in GBM progression by analyzing ICOSLG expression in both human glioma tissues and patient-derived GBM sphere cells (GSCs). We further examined its immune modulatory effects and the underlying molecular mechanisms. RESULTS: Bioinformatics analysis and GBM tissue microarray showed that upregulation of ICOSLG expression was associated with poor prognosis in patients with GBM. ICOSLG expression was upregulated preferentially in mesenchymal GSCs but not in proneural GSCs in a tumor necrosis factor-α/nuclear factor-kappaB-dependent manner. Furthermore, ICOSLG expression by mesenchymal GSCs promoted expansion of T cells that produced interleukin-10. Knockdown of the gene encoding ICOSLG markedly reduced GBM tumor growth in immune competent mice, with a concomitant downregulation of interleukin-10 levels in the tumor microenvironment. CONCLUSIONS: Inhibition of the ICOSLG-inducible co-stimulator axis in GBM may provide a promising immunotherapeutic approach for suppressing a subset of GBM with an elevated mesenchymal signature.

DOI： 10.1093/neuonc/noz204

PubMed

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J-GLOBAL

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Neuroendocrine tumors are rare, and little is known about the existence of cancer stem cells in this disease. Identification of the tumorigenic population will contribute to the development of effective therapies targeting neuroendocrine tumors. Surgically resected brain metastases from a primary neuroendocrine tumor of unknown origin were dissociated and cultured in serum-free neurosphere medium. Stem cell properties, including self-renewal, differentiation potential, and stem cell marker expression, were examined. Tumor formation was evaluated using intracranial xenograft models. The effect of temozolomide was measured in vitro by cell viability assays. We established the neuroendocrine tumor sphere cell line ANI-27S, which displayed stable exponential growth, virtually unlimited expansion in vitro, and expression of stem-cell markers such as CD133, nestin, Sox2, and aldehyde dehydrogenase. FBS-induced differentiation decreased Sox2 and nestin expression. On the basis of real-time PCR, ANI-27S cells expressed the neuroendocrine markers synaptophysin and chromogranin A. Intracranial xenotransplanted brain tumors recapitulated the original patient tumor and temozolomide exhibited cytotoxic effects on tumor sphere cells. For the first time, we demonstrated the presence of a sphere-forming, stem cell-like population in brain metastases from a primary neuroendocrine tumor. We also demonstrated the potential therapeutic effects of temozolomide for this disease.

DOI： 10.1007/s00795-017-0160-0

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Bacterial beta-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect beta-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri-and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing beta-galactosidase, and despite its efficiency in stainingwhole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional beta-galactosidase histochemistry. Here, we compare the traditional beta-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for beta-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri-and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of beta-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.

DOI： 10.1007/s12565-015-0300-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: GABA has important functions in brain plasticity related processes like memory, learning, locomotion and during the development of the nervous system. It is synthesized by the glutamic acid decarboxylase (GAD). There are two isoforms of GAD, GAD1 and GAD2, which are encoded by different genes. During embryonic development the transcription of GAD1 mRNA is regulated by alternative splicing and several alternative transcripts were distinguished in human, mouse and rat. Despite the fact that the structure of GAD1 gene has been extensively studied, knowledge of its exact structural organization, alternative promoter usage and splicing have remained incomplete.
Results: In the present study we report the identification and characterization of novel GAD1 splicing isoforms (GenBank: KM102984, KM102985) by analyzing genomic and mRNA sequence data using bioinformatics, cloning and sequencing. Ten mRNA isoforms are generated from GAD1 gene locus by the combined actions of utilizing different promoters and alternative splicing of the coding exons. Using RT-PCR we found that GAD1 isoforms share similar pattern of expression in different mouse tissues and are expressed early during development. Quantitative RT-PCR was used to investigate the expression of GAD1 isoforms and GAD2 in olfactory bulb, cortex, medial and lateral striatum, hippocampus and cerebellum of adult mouse. Olfactory bulb showed the highest expression of GAD1 transcripts. Isoforms 1/2 are the most abundant forms. Their expression is significantly higher in the lateral compared to the medial striatum. Isoforms 3/4, 5/6, 7/8 and 9/10 are barely detectable in all investigated regions except of the high expression in olfactory bulb. When comparing GAD1 expression with GAD2 we found that Isoforms 1/2 are the predominant isoforms. In situ hybridization confirmed the predominant expression of Isoforms 7/8 and 9/10 in the olfactory bulb and revealed their weak expression in hippocampus, cerebellum and some other areas known to express GAD1.
Conclusions: Generation of ten splicing isoforms of GAD1 was described including two so far uncharacterized transcripts. GAD1 splicing isoforms producing the shorter, enzymatically inactive GAD25 protein are expressed at very low level in adult mouse brain except in the olfactory bulb that is associated with neurogenesis and synaptic plasticity even during adulthood.

DOI： 10.1186/1471-2202-15-114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Ltd  

Since induced pluripotent stem (iPS) cells have differentiation potential into all three germ layer-derived tissues, efficient purification of target cells is required in many fields of iPS research. One useful strategy is isolation of desired cells from differentiated iPS cells by lineage-specific expression of a drug-resistance gene, followed by drug selection. With this strategy, we purified neural stem/ progenitor cells (NSCs), a good candidate source for regenerative therapy, from differentiated mouse iPS cells. We constructed a bicistronic expression vector simultaneously expressing blastici-din S resistance gene and DsRed under the control of tandem enhancer of a 257-base pair region of nestin second intron, an NSC-specific enhancer. This construct was efficiently inserted into the iPS genome by piggyBac transposon-mediated gene transfer, and the established subclone was differentiated into NSCs in the presence or absence of blasticidin S. Consequently, incubation with blas-ticidin S led to purification of NSCs from differentiated iPS cells. Our results suggest that a lineage-specific drug selection strategy is useful for purification of NSCs from differentiated iPS cells and that this strategy can be applied for the purification of other cell types. © AlphaMed Press 2013.

DOI： 10.5966/sctm.2012-0139

Scopus

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Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier Ltd  

DOI： 10.1016/B978-0-12-382219-2.00089-2

Scopus

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：関西医科大学医学会  

マウス脳を用いて、聴覚上行路各部位におけるコリン線維とムスカリン性受容体(m2、m3タイプ)の発現分布密度について測定した。その結果、聴覚上行路諸核において、これらコリン線維・受容体を介する情報転達・変換が、聴覚の伝達に一定の意義を有していることが示唆された。

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2012&ichushi_jid=J00279&link_issn=&doc_id=20121218420002&doc_link_id=10.5361%2Fjkmu.63.7&url=https%3A%2F%2Fdoi.org%2F10.5361%2Fjkmu.63.7&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In primate placenta, extravillous trophoblast (EVT) invades maternal tissue in temporally- and spatially-regulated fashions. We previously identified a novel placenta-specific cell-surface aminopeptidase, laeverin/aminopeptidase Q, which is expressed on EVT-lineage cells in the fetal membrane. Laeverin possesses a peptide-binding site that is evolutionally unique to primates, suggesting possible involvement of laeverin in a primate-specific phenomenon during placentation. Thus, this study was designed to elucidate the molecular characteristics and physiological roles of laeverin in human EVT.
Placental tissues of various developmental stages were subjected to immunostaining and western blotting. Effects of siRNA and a soluble form of recombinant laeverin on EVT cells isolated from primary villous explant cultures were examined using Matrigel invasion assays and cell proliferation assays.
Laeverin was specifically immunolocalized to HLA-G-positive EVT in placentas from early and term pregnancy. In primary villous explant cultures, laeverin expression was induced on the cell surface of the outgrowing EVT. In western blotting, laeverin protein was detected as two distinct bands at 130 and 160 kDa along with a broad band ranging from 200 to 270 kDa. De-glycosylation treatment showed that these native laeverin isotypes are N-linked glycoproteins sharing a common 115-kDa core protein. In invasion assays, the reduction of laeverin expression by siRNA suppressed migration of the isolated EVT, while the soluble form of recombinant laeverin enhanced its migration.
Laeverin is a specific cell-surface marker for human EVT and plays a regulatory role in EVT migration.

DOI： 10.1093/humrep/des068

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The GABA-synthesizing enzymes glutamate decarboxylase (GAD)1 and GAD2 are universally contained in GABAergic neurons in the central nervous system of the mouse and rat. The two isoforms are almost identically expressed throughout the brain and spinal cord. By using in situ hybridization, we found that the mouse lateral striatum concentrates medium-sized projection neurons with high-level expression of GAD1, but not of GAD2, mRNA. This was confirmed with several types of riboprobe, including those directed to the 5'-noncoding, 3'-noncoding and coding regions. Immunohistochemical localization of GAD1 also revealed predominant localization of the enzyme in the same striatal region. The lateral region of the mouse striatum, harboring such neurons, is ovoid in shape and extends between interaural +4.8 and +2.8, and at lateral 2.8 and dorsoventral 2.0. This intriguing region corresponds to the area that receives afferent inputs from the primary motor and sensory cortex that are presumably related to mouth and forelimb representations. The lateral striatum is included in the basal ganglia-thalamocortical loop, and is most vulnerable to various noxious stimuli in the neurodegeneration processes involving the basal ganglia. We have confirmed elevated expression of GAD1 mRNA, but not of GAD2 mRNA, also in the rat lateral striatum. Image analysis favored the view that the regional increase is caused by elevated cellular expression, and that the greatest number of medium-sized spiny neurons were positive for GAD1 mRNA. The GAD1 mRNA distribution in the mouse lateral striatum partially resembled those of GPR155 and cannabinoid receptor type 1 mRNAs, suggesting functional cooperation in some neurons.

DOI： 10.1111/j.1460-9568.2012.08001.x

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Publishing type：Research paper (scientific journal)  

DOI： 10.1002/ar.21186

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Cholinergic projections to auditory system are vital for coupling arousal with sound processing. Systematic search with in situ hybridization and immunohistochemistry indicated that the ventral nucleus of the medial geniculate body and the nucleus of the brachium of the inferior colliculus constituted cholinergic synaptic sites in the brainstem auditory system, containing a significant number of cholinergic axon terminals and m2 receptor-expressing cell bodies. Anat Rec, 293:1393-1399, 2010. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/ar.21186

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Emerging evidence suggests that GPR155, an integral membrane protein related to G-protein coupled receptors, has specific roles in Huntington disease and autism spectrum disorders. This study reports the structural organization of mouse GPR155 gene and the generation of five variants (Variants 1-5) of GPR155 mRNA, including so far unknown four variants. Further, it presents the level of expression of GPR155 mRNA in different mouse tissues. The mRNAs for GPR155 are widely expressed in adult mouse tissues and during development. In situ hybridization was used to determine the distribution of GPR155 in mouse brain. The GPR155 mRNAs are widely distributed in forebrain regions and have more restricted distribution in the midbrain and hindbrain regions. The highest level of expression was in the lateral part of striatum and hippocampus. The expression pattern of GPR155 mRNAs in mouse striatum was very similar to that of cannabinoid receptor type 1. The predicted protein secondary structure indicated that GPR155 is a 17-TM protein, and Variant 1 and Variant 5 proteins have an intracellular, conserved DEP domain near the C-terminal. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.05.162

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Human laeverin/aminopeptidase Q (LVRN/APQ) is a novel member of the M1 family of zinc aminopeptidases and is specifically expressed on the cell surface of human extravillous trophoblasts. Multiple sequence alignment of human M1 aminopeptidase revealed that the first Gly residue within the conserved exopeptidase motif of the M1 family, GXMEN motif, is uniquely substituted for His in human LVRN/APQ. In this study, we evaluated the roles of nonconserved His(379), comprising the exopeptidase motif in the enzymatic properties of human LVRN/APQ. We revealed that the substitution of His(379) with Gly caused significant changes in substrate specificity both toward fluorogenic substrates and natural peptide hormones. In addition, the susceptibilities of bestatin, a sensitive inhibitor for human LVRN/APQ, and natural inhibitory peptides were decreased in the H379G mutant. A molecular model suggested a conformational difference between wild-type and H379G human LVRN/APQs. These results indicate that His(379) of the enzyme plays essential roles in its distinctive enzymatic properties and contributes to maintaining the appropriate structure of the catalytic cavity of the enzyme. Our data may bring new insight into the biological significance of the unique exopeptidase motif of LVRN/APQ obtained during the evolution of primates.

DOI： 10.1074/jbc.M109.066712

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：耳鼻咽喉科ニューロサイエンス研究会  

マウスを用い、ムスカリン性アセチルコリン受容体のサブタイプm2(抑制性神経伝達受容体)、およびm3(興奮性神経伝達受容体)の発現を、コリン性入力線維(VAChT)の局在と比較した。VAChT免疫陽性瘤状線維は脚橋被蓋核、三叉神経運動核、顔面神経核、舌下神経核、迷走神経背側運動核において発現密度高値を示した。m2ムスカリン受容体mRNA(m2)も同様であったが、m3では発現細胞はほとんどなかった。VAChT免疫陽性線維は大脳皮質聴覚野、内側膝状体、下丘腕核、蝸牛神経腹側核において発現密度が高かった。m2は大脳皮質聴覚野、内側膝状体、下丘腕核・背側核・外側核、蝸牛神経背側核において発現密度が高かった。m3は大脳皮質聴覚野内側膝状体、下丘背側核、蝸牛神経背側核において発現密度が高かった。中枢聴覚路におけるVAChT、およびコリン受容体発現細胞におけるm2、m3の受容体の発現分布が判明し、聴覚障害に対するムスカリン受容体サブタイプに特異的な薬物の臨床応用が考えられた。

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

The interferon (IFN) is a paradigm of secretory protein. However, it has been poorly understood how its secretion is regulated in polarized epithelial cells. Recently, we had shown that exogenous IFNs transiently expressed in polarized monolayers were predominantly secreted to the side on which gene transfection had been performed, while stably expressed IFNs were secreted almost equally to the both cell sides. Since those modes of secretion did not affect each other, epithelial cell layers seemed to have at least two protein sorting/secretion pathways, one for transient expression and the other for stable expression, for identical secretory proteins. Furthermore, this dual secretion profile seemed to be mediated by distinct post-trans Golgi network vesicles, suggesting the involvement of lipid rafts in the sorting multiplicity. To address this issue, here we studied the effects of cholesterol depletion with methyl-beta-cyclodextrin (M beta CD) on the secretion profile of IFN-beta exogenously expressed in Madin-Darby canine kidney (MDCK) cells. The M beta CD-treatment, however, did not affect the profile in either transient or stable expression, although the architecture of zonula occuludin-1, which links to the tight junction, was substantially disrupted by the treatment. Further analysis of Triton X-100-insoluble cell extracts by sucrose density centrifugation demonstrated that IFN-beta was not apparently associated with lipid rafts in either transient or stable expression. These results suggest that lipid rafts may not be crucially involved in the regulation of secretion polarity of IFN-beta in the epithelial cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of the neurotransmitter acetylcholine. Seven types of mRNA for choline acetyltransferase that differ in the 5'-noncoding region are transcribed from the cholinergic gene locus from different promoter regions and produced by alternative splicing in the mouse. Digoxigenin-labeled riboprobes and in situ hybridization histochemistry were used to investigate the expression of N1, R1, R2, R3, R4 and total choline acetyltransferase mRNA in the mouse CNS. The relative levels of choline acetyltransferase transcripts differed dramatically in distinct subdivisions of the mature cholinergic nervous system. Neurons hybridizing with antisense riboprobes for all of the five investigated splice variants (R1, R2, R3, R4 and N1) as well as those hybridizing with riboprobe for the common protein-coding region were found in a number of expected regions in the CNS. They include the basal forebrain, striatum, pontomesencephalic tegmentum, motor and autonomic nuclei of the brainstem, and spinal cord. Neurons with a moderate to very high level of expression of R1 and R2 splice variants were distributed in both the forebrain and brainstem nuclei. On the other hand, R3, R4 and N1 splice variants revealed a moderate to high level of expression in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord compared to a low expression level in forebrain cholinergic structures. No expression of the N1, R1, R2, R3 and R4 splice variants was detectable in the neurons of the cerebral cortex, hippocampus and medial habenular nucleus. With the riboprobe for the common protein-coding region, the neurons of the medial habenular nucleus could be labeled at high level, while intrinsic cortical neurons were labeled at low level. Hippocampus revealed no significant hybridization for total choline acetyltransferase mRNA. These findings strongly suggested that: (1) R1 and R2 were the major splice variants expressed in the neurons of forebrain nuclei; (2) R1, R2, R3, R4 and N1 splice variants were almost equally expressed in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord; (3) inferring from a paucity of other isoforms, M type choline acetyltransferase mRNA is a splice variant predominantly expressed in the cerebral cortex and medial habenular nucleus. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2008.12.054

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

It is becoming evident that several aminopeptidases belonging to the M1 family such as aminopeptidase A (APA), placental leucine aminopeptidase (P-LAP), and adipocyte-derived leucine aminopeptidase (A-LAP) play important roles in the regulation of blood pressure under both the physiological and pathological conditions. They share HEXXH(X)(18)E zinc-binding and GAMEN motifs essential for enzymatic activities. In this review, the current situation regarding the biochemical characteristics of these enzymes including enzymatic properties and modes of action is summarized.

DOI： 10.1007/s10741-007-9064-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Laeverin/aminopeptidase Q (APQ) is a cell surface protein specifically expressed on human embryo-derived extravillous trophoblasts that invades the uterus during placentation. The cDNA cloning of Laeverin/APQ revealed that the sequence encodes a protein with 990 amino acid residues, and Laeverin/APQ contains the HEXXHX18E gluzincin motif, which is characteristic of the M1 family of aminopeptidases, although the exopeptidase motif of the family, GAMEN, is uniquely substituted for the HAMEN sequence. In this study, we expressed a recombinant human Laeverin/APQ using a baculovirus expression system, purified to homogeneity, and characterized its enzymatic properties. It was found that Laeverin/APQ had a broad substrate specificity toward synthetic substrate, although it showed a preference for Leu-4-methylcoumaryl-7-amide. Searching natural substrates, we found that Laeverin/ APQ was able to cleave the N-terminal amino acid of several peptides such as angiotensin III, kisspeptin-10, and endokinin C, which are abundantly expressed in the placenta. In contrast to the case with other M1 aminopeptidases, bestatin inhibited the aminopeptidase activity of Laeverin/ APQ much more effectively than other known aminopeptidase inhibitors. These results indicate that Laeverin/ APQis a novel bestatin-sensitive leucine aminopeptidase and suggest that the enzyme plays important roles in human placentation by regulating biological activity of key peptides at the embryo- maternal interface.

DOI： 10.1074/jbc.M702650200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2005.04.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

We have investigated the secretion polarity of human interferon-beta (HuIFN-beta) exogenously expressed in the porcine renal proximal tubule cell line, LLC-PK1,. In these cells, stably expressed HuIFN-beta was secreted to the apical and basolateral sides. However, when transiently expressed by apical lipofection, HuIFN-beta was secreted to both cell sides, while basolateral-preferential secretion was seen for basal transfection. Confocal imaging using enhanced green fluorescent protein (EGFP)-tagged HuIFN-beta revealed no difference in the subcellular distribution in either of the chimeric protein-expressing cells examined. These results suggest that the secretion polarity of HuIFN-beta is regulated by a post trans-Golgi network in a cell type-dependent manner.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon-beta (IFN-beta). Expression plasmids for human IFN-beta tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin-Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20degreesC) allowed DsRed2-tagged human IFN-beta to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocai microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins.

DOI： 10.1002/jcb.20203

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

We have recently demonstrated that when IFN-beta was exogenously expressed in epithelial cells, transiently expressed IFN-beta was predominantly secreted from the cell side to which the transfection was performed, while stably expressed one was almost equally secreted to the apical and basolateral sides. In the present study, we analyzed the subcellular transport of IFN-beta using confocal imaging with green fluorescent protein (GFP)-tagged IFN-beta in Madin-Darby canine kidney (MDCK) cells. Stably expressed and transiently expressed human IFN-beta (HuIFN-beta)-GFPs were seen in upper regions of the nucleus. In stable HuIFN beta-GFP-producing transformants, transiently expressed mouse IFN-beta (MuIFN-beta) was apparently co-localized with the bulk of the constitutive HuIFN beta-GFP proteins at TGN, and a significant quantity of them then appeared to pass into distinct post-TGN vesicles, accepting either type of IFN. Meanwhile, when cells were co-transfected with both expression vectors, transiently expressed both IFNs tended to co-localize not only at TGN but in post-TGN vesicles. These results suggest that stably and transiently expressed IFN-betas, albeit co-localized at TGN, were transported through apparently discriminated post-TGN routes.

DOI： 10.1002/jcp.20038

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

We have constructed a recombinant cDNA encoding the chimeric protein between human IFN-beta (HuIFN-beta) and enhanced green fluorescent protein (EGFP) to elucidate the intracellular localization of IFN-beta. Transient expression of the chimeric molecule, HuIFN-beta-EGFP, in L cells demonstrated that the chimeric molecule secreted from the cells had an intact biological activity as far as antiviral effect was concerned. Immunostaining of the transfected cells using anti-HuIFN-beta antibody demonstrated that green-fluorescence was co-localized with the IFN signal and its profile was similar to IFN signals in the cells transfected with HuIFN-beta expressing plasmid DNA. These results indicate that the HuIFN-beta-EGFP chimeric gene was expressed as a chimeric protein and the chimera was transported via the regular secretory pathway in the cells. In other cell types, the fluorescence derived from the chimeric protein was also seen on cytoplasmic vesicular structures. These results suggest that HuIFN-beta-EGFP will be a useful tool to investigate the intracellular trafficking processes of HuIFN-beta in a variety of cell types.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

We have evaluated and compared the efficacy of systemic administration of lipoplex formulations containing plasmids encoding IFN-beta or IFN-gamma, and a synthetic double-strand RNA poly I: poly C (pI: pC), a type I IFN inducer, in a lung metastasis model in which colon carcinoma CT-26 cells were inoculated intravenously into immunocompatible mice. Injection of lipoplexes containing plasmid DNA, regardless of IFN gene insertion, stimulated a transient increase in the serum concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and IFN-gamma, while injection of lipoplexes containing pI: pC led to a low level of TNF-alpha and undetectable IFN-gamma production. Furthermore, injection of these lipoplexes containing plasmids resulted in the production of a mixture of type I and type II IFNs, partly derived from the inserted IFN genes, in lung tissue cultures. In tumor-prophylactic experiments, intravenous injection of lipoplexes containing plasmid, regardless of IFN gene insertion, showed a significant reduction in lung metastatic nodules probably due to proinflammatory cytokines such as TNF-alpha and IFN-gamma nonspecifically induced by the CpG motifs in the plasmid and the type I IFNs produced. On the other hand, the antimetastatic effect of pI: pC-lipoplex seemed to be due mainly to IFN-beta induced by pI: pC. In established lung metastasis experiments, a single intravenous administration of lipoplexes containing IFN-beta gene or pI: pC, but not other lipoplexes, showed a significant therapeutic effect on the tumor metastasis: reduction in tumor nodules and prolongation of survival time of tumor-burden mice. The therapeutic effects were specifically impaired by anti-IFN-beta antibody treatment, indicating that IFN-beta produced by the lipoplexes played an important role in the suppression of established metastatic lung tumors. Thus, the local IFN-beta in the lung delivered by intravenous administration of lipoplex containing IFN-beta gene or pI: pC may be a convenient and useful method of inhibiting established metastatic lung tumors.

DOI： 10.1038/sj.cgt.7700617

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Epithelial cells are an attractive target for local gene delivery in gene therapy for which cytokine genes such as interferon (IFN) genes are promising. However, how the secretion of the gene products is regulated in epithelial cells has been insufficiently investigated. Here, we have studied the secretion polarity of IFN-beta expressed via gene transfection in mouse epithelial Pam-T cells on a bicameral culture system. In transient expression, IFN-beta was predominantly secreted from the cell membrane side on which the transfection was carried out. Meanwhile, the secretion of constitutive IFN-beta from stable transformants was apparently unpolarized. Interestingly, the transformants displayed a polarized secretion of transiently expressed IFN-beta in a transfection-side-dependent manner, their stable IFN-beta secretion remaining unpolarized. These results suggest that epithelial cells have at least dual protein sorting-secretion pathways, transient and stable, for the same secretory proteins, such as IFNs. (C) 2002 Elsevier Science (USA). All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

The effects of nitric oxide (NO) donors NOC5 [3-(2-hydroxy-1-(methylethyl)- 2-nitrosohydrazino)-1-propanamine] and NOC12 [N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine] on the permeability of 5(6)-carboxyfluorescein (CF) across the intestinal membrane were examined by an in vitro Ussing chamber method. The NO donors significantly increased the intestinal permeability of CF and their absorption-enhancing effects were concentration-dependent over the range of 0.01 to 0.1 mM. Regional differences in the absorption-enhancing effects of the NO donors were observed (colon. jejunum). The absorption-enhancing effect of NOC12 reduced as the molecular weights of compounds increased. Therefore, the degree of absorption-enhancing effect of NOC12 was dependent on the molecular weights of compounds. In the pretreatment studies with NOC12 and lactate dehydrogenase release studies, the absorption-enhancing effect of 0.1 mM NOC12 was reversible and less toxic to the colonic membrane. On the other hand, the absorption-enhancing effect of NOC12 was inhibited by the coadministration of 2-(4-carboxyphenyl) 4,4,5,5-tetramethylimidazole- 1-oxyl 3-oxide sodium salt, an NO scavenger, suggesting that NO can regulate the permeability of water-soluble drugs in the gut. Furthermore, NOC12 (0.1 and 1 mM) significantly decreased the transepithelial electrical resistance value of the colonic membrane, suggesting that the absorption-enhancing mechanism of NOC12 may be partly related to the dilation of the tight junction in the epithelium via a paracellular route. These findings suggest that NO donors may be useful to enhance the intestinal absorption of poorly absorbable drugs.

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2011.07.605

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2011.07.1443

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Language：English   Publisher：日本神経化学会  

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.2224

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publisher：(一社)日本解剖学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2009.09.389

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2009.09.267

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J-GLOBAL

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Language：Japanese  

CiNii Article

CiNii Books

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Language：Japanese  

CiNii Article

CiNii Books

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Event date： 2019.12.9 - 2019.12.12

Presentation type：Oral presentation (general)  

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Venue：神戸  

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Venue：岐阜  

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Venue：神戸  

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Venue：長崎  

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Venue：郡山  

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Venue：下野  

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Venue：生駒  

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Venue：高松  

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Venue：横浜  

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Venue：甲府  

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Venue：甲府  

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Venue：西宮  

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Venue：神戸  

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Venue：名古屋  

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Venue：岡山  

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Venue：神戸  

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Venue：大阪  

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Venue：大阪  

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Venue：大阪  

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Venue：横浜、日本  

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Presentation type：Symposium, workshop panel (nominated)  

Venue：静岡、日本  

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Presentation type：Symposium, workshop panel (nominated)  

Venue：横浜、日本  

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Venue：横浜、日本  

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Venue：Patras, Greece  

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Venue：大阪、日本  

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Venue：Kanagawa, Japan  

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Presentation type：Symposium, workshop panel (nominated)  

Venue：静岡、日本  

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Venue：仙台、日本  

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Venue：東京、日本  

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Venue：京都、日本  

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Venue：Kyoto, Japan  

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Venue：Kyoto, Japan  

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Venue：京都、日本  

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Venue：京都、日本  

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Venue：長崎、日本  

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Venue：Michigan, USA  

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Venue：千葉、日本  

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Venue：Seoul, Korea  

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Venue：千葉、日本  

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Venue：札幌、日本  

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Presentation type：Symposium, workshop panel (nominated)  

Venue：京都、日本  

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Venue：San Francisco, USA  

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