Yuka Hiroshima, Rie Kido, Jun-ichi Kido, Mika Bandou, Kaya Yoshida, Akikazu Murakami and Yasuo Shinohara : Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system, Odontology, 2024.
(Tokushima University Institutional Repository: 119164)
2.
Takeshi Ito, Sayaka Kajita, Minori Fujii and Yasuo Shinohara : Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase, Microbiology Spectrum, Vol.11, No.3, e0016823, 2023.
(Summary)
The emergence of drug-resistant variants of malaria-causing Plasmodium parasites is a life-threatening problem worldwide. Investigation of the physiological function of individual parasite proteins is a prerequisite for a deeper understanding of the metabolic pathways required for parasite survival and therefore a requirement for the development of novel antimalarials. A Plasmodium membrane protein, malate-quinone oxidoreductase (MQO), is thought to contribute to the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) and is an antimalarial drug target. However, there is little information on its expression and function. Here, we investigated the function of Plasmodium falciparum MQO (PfMQO) in mitochondria using a yeast heterologous expression system. Using a yeast deletion mutant of mitochondrial malate dehydrogenase (MDH1), which is expected to be functionally similar to MQO, as a background strain, we successfully constructed PfMQO-expressing yeast. We confirmed that expression of PfMQO complemented the growth defect of the MDH1 deletion, indicating that PfMQO can adopt the metabolic role of MDH1 in energy transduction for growth in the recombinant yeast. Analysis of cell fractions confirmed that PfMQO was expressed and enriched in yeast mitochondria. By measuring MQO activity, we also confirmed that PfMQO expressed in yeast mitochondria was active. Measurement of oxygen consumption rates showed that mitochondrial respiration was driven by the TCA cycle through PfMQO. In addition, we found that MQO activity was enhanced when intact mitochondria were sonicated, indicating that the malate binding site of PfMQO is located facing the mitochondrial matrix.
Jun-ichi Kido, Yuka Hiroshima, Rie Kido, Kaya Yoshida, Yuji Inagaki, Koji Naruishi, Kazuaki Kajimoto, Masatoshi Kataoka, Yasuo Shinohara and Hiromichi Yumoto : Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells., Journal of Periodontal Research, Vol.58, No.2, 262-273, 2023.
Yuka Hiroshima, Jun-ichi Kido, Rie Kido, Kaya Yoshida, Mika Bandou, Kazuaki Kajimoto, Hiromichi Yumoto and Yasuo Shinohara : β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells., Odontology, Vol.111, 830-838, 2023.
Kazuto Takegawa, Takeshi Ito, Atsushi Yamamoto, Naoshi Yamazaki, Mitsuru Shindo and Yasuo Shinohara : KH-17, a simplified derivative of bongkrekic acid, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane, Chemical Biology & Drug Design, Vol.101, No.4, 865-872, 2023.
(Summary)
Two natural products, bongkrekic acid and carboxyatractyloside, are known to specifically inhibit the mitochondrial ADP/ATP carrier from its matrix side and cytosolic side, respectively, in concentration ranges of 10-6 M. In the present study, we investigated the manner of action of a synthetic bongkrekic acid derivative, KH-17, lacking three methyl groups, one methoxy group, and five internal double bonds, on the mitochondrial ADP/ATP carrier. At slightly acidic pH, KH-17 inhibited mitochondrial [3 H]ADP uptake, but its inhibitory action was about 10 times weaker than that of its parental compound, bongkrekic acid. The main site of action of KH-17 was confirmed as the matrix side of the ADP/ATP carrier by experiments using submitochondrial particles, which have an inside-out orientation of the inner mitochondrial membrane. However, when we added KH-17 to mitochondria at neutral pH, it had a weak inhibitory effect on [3 H]ADP uptake, and its inhibitory strength was similar to that of bongkrekic acid. These results indicated that KH-17 weakly inhibits the ADP/ATP carrier not only from the matrix side but also from the cytosolic side. To ascertain whether this interpretation was correct, we examined the effects of KH-17 and carboxyatractyloside on mitochondrial [3 H]ADP uptake at two [3 H]ADP concentrations. We found that both KH-17 and carboxyatractyloside showed a stronger inhibitory effect at the lower [3 H]ADP concentration. Therefore, we concluded that the bongkrekic acid derivative, KH-17, weakly inhibits the mitochondrial ADP/ATP carrier from both sides of the inner mitochondrial membrane. These results suggested that the elimination of three methyl groups, one methoxy group, and five internal double bonds present in bongkrekic acid altered its manner of action towards the mitochondrial ADP/ATP carrier. Our data will help to improve our understanding of the interaction between bongkrekic acid and the mitochondrial ADP/ATP carrier.
Yoshinobu Fujiwara, Takeshi Ito, Atsumi Toiyama, Takenori Yamamoto, Naoshi Yamazaki, Mitsuru Shindo and Yasuo Shinohara : Suramin Inhibits Mitochondrial ADP/ATP Carrier, Not Only from the Cytosolic Side But Also from the Matrix Side, of the Mitochondrial Inner Membrane, BPB Reports, Vol.4, No.3, 92-97, 2021.
(Summary)
<p>Suramin was earlier reported to show inhibitory effects on the mitochondrial ADP/ATP carrier. However, two important questions, i) whether it shows a specific inhibition of the ADP/ATP carrier when applied to isolated mitochondria, and ii) whether it inhibits the mitochondrial ADP/ATP carrier only from the cytosolic side or from the matrix side, as has been observed with its canonical inhibitors of carboxyatractyloside or bongkrekic acid, remain to be answered. In the present study, we sought exact answers to these questions. As for the first question, suramin showed certain inhibitory effects on the mitochondrial respiratory chain; and at a concentration of 25 μM it showed strong inhibition of the mitochondrial ADP/ATP carrier. This property was due to its weaker inhibitory effects on the mitochondrial ADP/ATP carrier than those of carboxyatractyloside or bongkrekic acid. As for the second question, suramin inhibited the ADP/ATP carrier from both sides of the mitochondrial inner membrane. Thus, suramin was concluded to be utilizable as a new type of inhibitor for the ADP/ATP carrier; but we must pay attention to its side-effects, especially when it is applied to whole mitochondria.</p>
Takenori Yamamoto, Mizune Ozono, Akira Watanabe, Kosuke Maeda, Atsushi Nara, Mei Hashida, Yusuke Ido, Yuka Hiroshima, Akiko Yamada, Hiroshi Terada and Yasuo Shinohara : Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake, Biochimica et Biophysica Acta (BBA) - Bioenergetics, 148061, 2019.
(Summary)
The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca-uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca-uptake function. Thus, it is essential for the Ca uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca-uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU.
Naoshi Yamazaki, Keisuke Kanazawa, Maria Kimura, Hironobu Ike, Makiko Shinomiya, Shouko Tanaka, Yasuo Shinohara, Noriaki Minakawa, Kouji Itou and Yoshiharu Takiguchi : Use of modified U1 small nuclear RNA for rescue from exon 7 skipping caused by 5-splice site mutation of human cathepsin A gene, Gene, Vol.677, 41-48, 2018.
(Summary)
Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called galactosialidosis. In a certain number of galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence galactosialidosis. This mutation seems to make the interaction between the 5'-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5'-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene.
Takenori Yamamoto, Moe Tsunoda, Mizune Ozono, Akira Watanabe, Kazumasa Kotake, Yuka Hiroshima, Akiko Yamada, Hiroshi Terada and Yasuo Shinohara : Polyethyleneimine renders mitochondrial membranes permeable by interacting with negatively charged phospholipids in them, Archives of Biochemistry and Biophysics, 2018.
(Summary)
Polyethyleneimines (PEIs) are used for transfection of cells with nucleic acids. Meanwhile, the interaction of PEI with mitochondria causes cytochrome c release prior to apoptosis; the mechanisms how PEI causes this permeabilization of mitochondrial membranes and the release of cytochrome c remain unclear. To clarify these mechanisms, we examined the effects of branched-type PEI and linear-type PEI, each of which was 25 kDa in size, on mitochondria. The permeabilization potency of mitochondrial membranes by branched PEI was stronger than that by linear PEI. The permeabilization by PEIs were insensitive to permeability-transition inhibitors, indicating that PEI-induced permeabilization was not attributed to permeability transition. Meanwhile, PEIs caused permeabilization of artificial lipid vesicles; again, the permeabilization potency of branched PEI was stronger than that of linear PEI. Such a difference in this potency was close to that in the case of isolated mitochondria, signifying that the PEI-induced permeabilization of mitochondrial membranes could be attributed to PEI's interaction with the phospholipid phase. Furthermore, this PEI-induced permeabilization of the lipid vesicles was observed only in the case of lipid vesicles including negatively charged phospholipids. These results indicate that PEIs interacted with negatively charged phospholipids in the mitochondrial membranes to directly lead to their permeabilization.
Yuka Hiroshima, Takenori Yamamoto, Masahiro Watanabe, Yoshinobu Baba and Yasuo Shinohara : Effects of cold exposure on metabolites in brown adipose tissue of rats., Molecular Genetics and Metabolism Reports, Vol.15, 36-42, 2018.
(Summary)
Brown adipose tissue (BAT) plays an important role in regulation of energy expenditure while adapting to a cold environment. BAT thermogenesis depends on uncoupling protein 1 (UCP1), which is expressed in the inner mitochondrial membranes of BAT. Gene expression profiles induced by cold exposure in BAT have been studied, but the metabolomic biological pathway that contributes to the activation of thermogenesis in BAT remains unclear. In this study, we comprehensively compared the relative levels of metabolites between the BAT of rats kept at room temperature (22 °C) and of those exposed to a cold temperature (4 °C) for 48 h using capillary electrophoresis (CE) time-of-flight mass spectrometry (TOFMS) and liquid chromatography (LC)-TOFMS. We identified 218 metabolites (137 cations and 81 anions) by CE-TOFMS and detected 81 metabolites (47 positive and 34 negative) by LC-TOFMS in BAT. We found that cold exposure highly influenced the BAT metabolome. We showed that the cold environment lead to lower levels of glycolysis and gluconeogenesis intermediates and higher levels of the tricarboxylic acid (TCA) cycle metabolites, fatty acids, and acyl-carnitine metabolites than control conditions in the BAT of rats. These results indicate that glycolysis and β-oxidation of fatty acids in BAT are positive biological pathways that contribute to the activation of thermogenesis by cold exposure, thereby facilitating the generation of heat by UCP1. These data provide useful information for understanding the basal metabolic functions of BAT thermogenesis in rats in response to cold exposure.
Yuka Hiroshima, Eijiro Sakamoto, Kaya Yoshida, Kaori Abe, Koji Naruishi, Takenori Yamamoto, Yasuo Shinohara, Jun-ichi Kido and Carolyn L Geczy : Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells., Journal of Cellular Biochemistry, Vol.119, No.2, 1591-1603, 2018.
(Summary)
Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE + PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis. This article is protected by copyright. All rights reserved.
Hirokazu Miyoshi, Mitsunori Hiroura, Kazunori Tsujimoto, Namiko Irikura, Tamaki Otani and Yasuo Shinohara : Preparation of new scintillation imaging material composed of scintillator-silica fine powders and its imaging of tritium, Radiation Protection Dosimetry, Vol.174, No.4, 478-484, 2017.
(Summary)
A new scintillation imaging material [scintillator-silica fine powder (FP)] was prepared using silica FPs and scintillator-encapsulating silica nanoparticles (NPs) (scintillator-silica NPs). The wt% values of scintillator-silica NPs on the scintillator-silica FPs were 38, 43, 36 and 44%. Scintillation images of 3H, 63Ni, 35S, 33P, 204Tl, 89Sr and 32P dropped on the scintillator-silica FPs were obtained at about 37 kBq per 0.1-10 µl with a charge-coupled device (CCD) imager for a 5 min exposure. In particular, high-intensity CCD images of 35S were selectively obtained using the 2.25, 4.77 and 10 µm silica FPs with scintillator-silica NPs owing to the residual S of dimethyl sulfoxide in the preparation. Scintillation images of 3H at 1670 ± 9 Bq/0.5 µl and 347 ± 6 Bq/0.5 µl dropped in a 2 mm hole on the scintillator-silica FPs (6.78 and 10 µm) were also obtained using the CCD imager for a 2 h exposure.
Kaya Yoshida, Hirohiko Okamura, Yuka Hiroshima, Kaori Abe, Jun-ichi Kido, Yasuo Shinohara and Kazumi Ozaki : PKR induces the expression of NLRP3 by regulating the NF-κB pathway in Porphyromonas gingivalis-infected osteoblasts, Experimental Cell Research, Vol.354, No.1, 57-64, 2017.
(Summary)
The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases. In this study, we investigated the effects of PKR on NLRP3 expression and NF-κB activity in P. gingivalis infected osteoblasts. We first constructed a SNAP26b-tagged P.gingivalis (SNAP-P. g.) and traced its internalization into the cell. SNAP-P. g. increased the activity of PKR and NF-κB and also induced NLRP3 expression in osteoblasts. Inhibition of NF-κB attenuated SNAP-P. g.-induced NLRP3 expression. The knockdown of PKR using shRNA decreased both the activity of NF-κB and the expression of NLRP3 induced by SNAP-P.g.. We therefore concluded that in osteoblasts, P. gingivalis activated PKR, which in turn increased NLRP3 expression by activating NF-κB. Our results suggest that PKR modulates inflammation by regulating the expression of the NLRP3 inflammasome through the NF-κB pathway in periodontal diseases.
R Sugahara, A Jouraku, T Nakakura, M Minaba, Takenori Yamamoto, Yasuo Shinohara, H Miyoshi and T Shiotsuki : Tissue-specific expression and silencing phenotypes of mitochondrial phosphate carrier paralogues in several insect species., Insect Molecular Biology, 2017.
(Summary)
The mitochondrial phosphate carrier gene (PiC) encodes a membrane protein that mediates the supply of inorganic phosphate from the cytosol into the mitochondrial matrix. This substrate-specific transport system plays an important role in efficient ATP synthesis. Mammals appear to have only one PiC with two alternative splicing variants whose functional differences remain unclear. The present study is the first to characterize the multiple genes that encode PiC in insects. Bombyx mori was found to have two PiC paralogues, one ubiquitous and one testis-specific, the latter seeming to be present only in Lepidoptera. Drosophila melanogaster was found to harbour two PiC paralogues, whereas Liriomyza chinensis, another dipteran, has three PiC paralogues. Two PiCs were found to be present in Plautia stali, and silencing either of these genes affected the normal development of P. stali nymphs, although their expression patterns differed amongst tissues. Schistocerca gregaria and Locusta migratoria have two PiC each, with different expression patterns. Tribolium castaneum was found to have only one PiC, which appears to play an essential role in larval development. Thus, although the inorganic phosphate transport system appears to be conserved across eukaryotes, PiC has become specialized in the different tissues of different insect species.
Yuya Yoshimura, Kosuke Fujisaki, Takenori Yamamoto and Yasuo Shinohara : Pharmacokinetic Studies of Orally Administered Magnesium Oxide in Rats., Journal of the Pharmaceutical Society of Japan, 2017.
(Summary)
Magnesium oxide (MgO) tablets are widely used as laxatives in patients with constipation. Recently, the "Revision of Precautions on the Use of Magnesium Oxide" has been issued by the Japanese Pharmaceuticals and Medical Devices Agency, warning against the risk of hypermagnesemia with the use of MgO. However, the majority of physicians continue to administer MgO for constipation without adequately considering its safe use. In the present study, we performed two analyses using an identical lot of MgO tablets and evaluated the risk of hypermagnesemia. Approximately 90% of the MgO tablets dissolved within 120 min in dissolution testing; it was believed to form an absorbable state for magnesium. With orally administered MgO, 15% is absorbed in the body and 85% is excreted via the feces without being detected in pharmacokinetic analysis. Magnesium absorbed into the plasma demonstrated peak concentration 3 h after administration and was excreted via the urine within 48 h.
The role of voltage-dependent anion channel (VDAC) as a metabolic gate of the mitochondrial outer membrane has been firmly established; however, its involvement in the regulation of mitochondrial permeability transition (PT) remains extremely controversial. Although some low-molecular-weight chemicals have been proposed to modulate the regulatory role of VDAC in the induction of PT, direct binding between these chemicals and VDAC has not yet been demonstrated. In the present study, we investigated whether the ubiquinone molecule directly binds to VDAC in Saccharomyces cerevisiae mitochondria through a photoaffinity labeling technique using two photoreactive ubiquinones (PUQ-1 and PUQ-2). The results of the labeling experiments demonstrated that PUQ-1 and PUQ-2 specifically bind to VDAC1 and that the labeled position is located in the C-terminal region Phe221-Lys234, connecting the 15th and 16th -strand sheets. Mutations introduced in this region (R224A, Y225A, D228A, and Y225A/D228A) hardly affected the binding affinity of PUQ-1. PUQ-1 and PUQ-2 both significantly suppressed the Ca(2+)-induced mitochondrial PT (monitored by mitochondrial swelling) at the one digit µM level. Thus, the results of the present study provided, for the first time to our knowledge, direct evidence indicating that the ubiquinone molecule specifically binds to VDAC1 through its quinone-head ring.
Namiko Irikura, Hirokazu Miyoshi and Yasuo Shinohara : Scintillation imaging of tritium radioactivity distribution during tritiated thymidine uptake by PC12 cells using a melt-on scintillator, Applied Radiation and Isotopes, Vol.120, 11-16, 2016.
(Summary)
A scintillation image of tritium fixed in a melt-on scintillator was obtained using a charged-coupled device (CCD) imager, and a linear relationship was observed between the intensity of the scintillation image and the radioactivity of tritium. In a [(3)H]thymidine uptake experiment, a linear correlation between the intensity of the CCD image and the dilution ratio of cells was confirmed. Scintillation imaging has the potential for use in direct observation of tritium radioactivity distribution.
Ryohei Yamagoshi, Takenori Yamamoto, Mitsuru Hashimoto, Ryohei Sugahara, Takahiro Shiotsuki, Hideto Miyoshi, Hiroshi Terada and Yasuo Shinohara : Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning., Mitochondrion, Vol.32, 1-9, 2016.
(Summary)
The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (NrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, NrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that NrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated NrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of NrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of NhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria.
Takenori Yamamoto, Ryohei Yamagoshi, Kazuki Harada, Mayu Kawano, Naoki Minami, Yusuke Ido, Kana Kuwahara, Atsushi Fujita, Mizune Ozono, Akira Watanabe, Akiko Yamada, Hiroshi Terada and Yasuo Shinohara : Analysis of the structure and function of EMRE in a yeast expression system., Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1857, No.6, 831-839, 2016.
(Summary)
The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.
Keita Takagi, Takashi Ohgita, Takenori Yamamoto, Yasuo Shinohara and Kentaro Kogure : Transmission of external environmental pH information to the inside of liposomes via pore-forming proteins embedded within the liposomal membrane, Chemical & Pharmaceutical Bulletin, Vol.64, No.5, 432-438, 2016.
(Summary)
Liposomes are closed-membrane vesicles comprised of lipid bilayers, in which the inside of the vesicles is isolated from the external environment. Liposomes are therefore often used as models for biomembranes and as drug delivery carriers. However, materials encapsulated within liposomes often cannot respond to changes in the external environment. The ability of enclosed materials to maintain their responsiveness to changes in the external environment following encapsulation into liposomes would greatly expand the applicability of such systems. We hypothesize that embedding pore-like "access points" into the liposomal membrane could allow for the transmission of information between the internal and external liposomal environments and thus overcome this inherent limitation of conventional liposomes. To investigate this, we evaluated whether a change in the pH of an external solution could be transmitted to the inside of liposomes through the pore-forming protein, yeast voltage-dependent anion channel (VDAC). Transmission of a pH change via VDAC was evaluated using a polyglutamic acid/doxorubicin complex (PGA/Dox) as an internal pH sensor. Upon encapsulation into conventional liposomes, PGA/Dox exhibits no pH sensitivity due to isolation from the external environment. On the other hand, PGA/Dox was found to retain its pH sensitivity upon encapsulation into VDAC-reconstituted liposomes, suggesting that VDAC facilitated the transmission of information on the pH of the external environment to the inside of the liposomes. In conclusion, we successfully demonstrated the transmission of information between the external and internal liposomal environments by a stable pore-like structure embedded into the liposomal membranes, which serve as access points.
Atsushi Yamamoto, Keisuke Hasui, Hiroshi Matsuo, Katsuhiro Okuda, Masato Abe, Kenji Matsumoto, Kazuki Harada, Yuya Yoshimura, Takenori Yamamoto, Kazuto Ohkura, Mitsuru Shindo and Yasuo Shinohara : Bongkrekic acid analogue, lacking one of the carboxylic groups of its parent compound, shows moderate but pH-insensitive inhibitory effects on the mitochondrial ADP/ATP carrier., Chemical Biology & Drug Design, Vol.86, No.5, 1304-1322, 2015.
(Summary)
Bongkrekic acid (BKA), isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In the present study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ATP carrier. Rough screening of these chemicals, done by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, 7, 16, and 17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of BKA for its pH- dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ATP carrier was also clearly demonstrated. This article is protected by copyright. All rights reserved.
Ryohei Sugahara, Akiya Jouraku, Takayo Nakakura, Takahiro Kusakabe, Takenori Yamamoto, Yasuo Shinohara, Hideto Miyoshi and Takahiro Shiotsuki : Two Adenine Nucleotide Translocase Paralogues Involved in Cell Proliferation and Spermatogenesis in the Silkworm Bombyx mori., PLoS ONE, Vol.10, No.3, e0119429, 2015.
(Summary)
Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4.
Kana Kuwahara, Kazuki Harada, Ryohei Yamagoshi, Takenori Yamamoto and Yasuo Shinohara : Effects of employment of distinct strategies to capture antibody on antibody delivery into cultured cells., Molecular and Cellular Biochemistry, Vol.404, No.1, 25-30, 2015.
(Summary)
The characteristics of antibody delivery into cultured HeLa cells were examined using two delivery systems. Both systems used a cell-penetrating peptide as a tool for intrusion of an antibody into the cells, but either a "protein A derivative" or "hydrophobic motif" was employed to capture the antibody. When we examined the uptake of the Alexa Fluor-labeled antibody by the use of these two systems, both systems were found to effectively deliver the antibody into the cultured cells. However, when we compared the amount of antibody delivered by these systems with the amount of transferrin uptake, the former was 10 times smaller than the latter. The lower efficiency of antibody delivery than transferrin uptake seemed to be attributable to the involvement of the antibody delivery reagent, which failed to catch the antibody molecule. This interpretation was validated by an experiment using a larger amount of antibody, and the amount of antibody delivered by the "protein A derivative" system under this condition was determined to be 13 ng proteins/10(5) cells. The antibody delivery achieved by the "protein A derivative" or "hydrophobic motif" showed two differences, i.e., a difference in intracellular distribution of the delivered antibody molecules and a difference in the fluorescence spectrum observed with cellular lysates. Possible reasons for these differences between the two delivery systems are discussed.
Takenori Yamamoto, Taisuke Matsuo, Atsushi Yamamoto, Ryohei Yamagoshi, Kazuto Ohkura, Masatoshi Kataoka and Yasuo Shinohara : Immunoblotting with Peptide Antibodies: Differential Immunoreactivities Caused by Certain Amino Acid Substitutions in a Short Peptide and Possible Effects of Differential Refolding of the Peptide on a Nitrocellulose or PVDF Membrane., Methods in Molecular Biology, Vol.1348, 303-310, 2015.
(Summary)
Immunodetection using antibodies, e.g., Western blotting, is generally utilized to measure the amount of a certain protein in a protein mixture. For valid interpretation of results observed by immunodetection, strict attention must be paid to the factors affecting the immunoreactivities of the antibodies. We here describe the step-by-step procedures to demonstrate that substitution of certain amino acids in a peptide can cause remarkable differences in its immunoreactivity with antibodies against epitope tags in the immobilized peptide. Refolding of the peptide on the membrane in a way that masks the epitope to different degrees was the possible reason for their distinct immunoreactivities with the antibodies. The results in this chapter suggest that we need to interpret carefully the experimental results involving immunodetection.
Takuya Suga, Yukihiro Asami, Shohei Hashimoto, Kenichi Nonaka, Masato Iwatsuki, Takuji Nakashima, Yoshihiro Watanabe, Ryohei Sugahara, Takahiro Shiotsuki, Takenori Yamamoto, Yasuo Shinohara, Naoya Ichimaru, Masatoshi Murai, Hideto Miyoshi, Satoshi Ōmura and Kazuro Shiomi : Trichopolyn VI: a new peptaibol insecticidal compound discovered using a recombinant Saccharomyces cerevisiae screening system., The Journal of General and Applied Microbiology, Vol.61, No.3, 82-87, 2015.
(Summary)
In the course of searching for insecticides from soil microorganisms, we found that a fermentation broth of the fungus, Trichoderma brevicompactum FKI-6324, produced Trichopolyn VI, a new peptaibol, which possessed significant insecticidal potential. Spectroscopic analysis showed the compound to be a new trichopolyn I derivative. This paper describes the isolation, structure elucidation and biological activity of trichopolyn VI.
Takenori Yamamoto, Mika Ito, Keita Kageyama, Kana Kuwahara, Kikuji Yamashita, Yoshiharu Takiguchi, Seiichiro Kitamura, Hiroshi Terada and Yasuo Shinohara : Mastoparan peptide causes mitochondrial permeability transition not by interacting with specific membrane proteins but by interacting with the phospholipid phase., The FEBS Journal, Vol.281, No.17, 3933-3944, 2014.
(Summary)
The mastoparan peptide is known as an inducer of the mitochondrial permeability transition. Although mastoparan was suggested to interact with a proteinaceous target in mitochondria to induce this transition, the action sites of mastoparan have not yet been investigated. To clarify whether specific interactions of mastoparan with receptors or enzymes are associated with the induction of this permeability transition, we examined the effects of d-isomeric peptides, which were synthesized using d-amino acids assembled in endogenous (inverso mastoparan) and reverse (retro-inverso mastoparan) orientations. When we added inverso mastoparan to isolated mitochondria, the peptide caused the permeability transition in a partially cyclosporin A-sensitive manner at lower doses and in a cyclosporin A-insensitive manner at higher ones. The manners of action and the potencies of inverso mastoparan were close to those of parent mastoparan, indicating that the targets of mastoparan for induction of the permeability transition were neither receptors, nor enzymes in the mitochondria. Retro-inverso mastoparan also had the same effect on the mitochondria as mastoparan, although the potencies of the effect were weaker. Not only on mitochondria, but also on phospholipid vesicles, mastoparan and inverso mastoparan showed massive permeabilization effects at the same potencies, although retro-inverso mastoparan showed weaker ones. These results indicate that mastoparan interacted with the phospholipid phase of the mitochondrial membrane (and not with specific proteins) to induce the permeabilization in cyclosporin A-sensitive and -insensitive manners.
(Keyword)
Animals / Cyclosporine / Male / Membrane Lipids / Mitochondria, Liver / Mitochondrial Membrane Transport Proteins / Mitochondrial Membranes / Peptides / Phosphatidylcholines / Rats / Stereoisomerism / Wasp Venoms
Naoto Okada, Takeshi Hanafusa, Takumi Sakurada, Kazuhiko Teraoka, Toshihide Kujime, Masahiro Abe, Yasuo Shinohara, Kazuyoshi Kawazoe and Kazuo Minakuchi : Risk Factors for Early-Onset Peripheral Neuropathy Caused by Vincristine in Patients With a First Administration of R-CHOP or R-CHOP-Like Chemotherapy, Journal of Clinical Medicine Research, Vol.6, No.4, 252-260, 2014.
(Summary)
Peripheral neuropathy is a well-known side effect of vincristine (VCR), a microtubule inhibitor used for R-CHOP or R-CHOP-like (namely R-CVP and R-THP-COP) regimens. Previous studies have shown that both the total dose of VCR and the number of treatment cycles are related to the incidence of VCR-induced peripheral neuropathy (VIPN). However, VIPN will also occur during the first treatment cycle regardless of the total dose of VCR or number of treatment cycles (early-onset VIPN). There is little information about early-onset VIPN, and it is difficult to predict. The present study's goal was to identify risk factors for early-onset VIPN. We analyzed the case records of patients who had their first administration of an R-CHOP or R-CHOP-like regimen between April 2008 and August 2013 at Tokushima University Hospital in Tokushima, Japan. To identify the risk factors for early-onset VIPN, we performed univariate and multivariate logistic regression analyses. Forty-one patients underwent an R-CHOP or R-CHOP-like regimen for the first time at Tokushima University Hospital between April 2008 and August 2013, and 14 patients had grade 1 or higher early-onset VIPN. A univariate analysis revealed that age, the dose of VCR and the concomitant use of aprepitant appeared to be the risk factors of early-onset VIPN. In our calculation using receiver-operator characteristics curves, the cut-off value for patient age was 65 years and that of the dose of VCR was 1.9 mg. A multivariate analysis revealed that VCR dose ≥ 1.9 mg and the concomitant use of the antiemetic aprepitant were independent risk factors for early-onset VIPN. Our present study showed that the patients who had VCR dose ≥ 1.9 mg and the concomitant use of aprepitant had the risk for early-onset VIPN. This suggests that it is important to use aprepitant in light of the risk of early-onset VIPN and the benefit of aprepitant's antiemetic effect in R-CHOP and R-CHOP-like regimens.
Yusuke Ido, Tatsuki Yoshitomi, Kazuto Ohkura, Takenori Yamamoto and Yasuo Shinohara : Utility of syntenic relationships of VDAC1 pseudogenes for not only an understanding of the phylogenetic divergence history of rodents, but also ascertaining possible pseudogene candidates as genuine pseudogenes., Genomics, Vol.104, No.2, 128-133, 2014.
(Summary)
Rodent and human genomes were screened to identify pseudogenes of the type 1 voltage-dependent anion channel (VDAC1) in mitochondria. In addition to the 16 pseudogenes of rat VDAC1 identified in our recent study, 15 and 13 sequences were identified as pseudogenes of VDAC1 in mouse and human genome, respectively; and 4, 2, and 1 sequences, showing lower similarities with the VDAC1 sequence, were identified as "possible pseudogene candidates" in rat, mouse, and human, respectively. No syntenic combination was observed between rodent and human pseudogenes, but 2 and 1 possible pseudogene candidates of VDAC1 of rat and mouse, respectively, were found to have syntenic counterparts in mouse and rat genome, respectively; and these syntenic counterparts were genuine VDAC1 pseudogenes. Therefore, syntenic combinations of pseudogenes of VDAC1 were useful not only for a better understanding of the phylogenetic divergence history of rodents but also for ascertaining possible pseudogene candidates as genuine pseudogenes.
Takuya Hada, Takenori Yamamoto, Atsushi Yamamoto, Kazuto Ohkura, Naoshi Yamazaki, Yoshiharu Takiguchi and Yasuo Shinohara : Comparison of the catalytic activities of three isozymes of carnitine palmitoyltransferase 1 expressed in COS7 cells, Applied Biochemistry and Biotechnology, Vol.172, No.3, 1486-1496, 2014.
(Summary)
The enzyme carnitine palmitoyltransferase 1 (CPT1) catalyzes the transfer of an acyl group from acyl-CoA to carnitine to form acylcarnitine, and three isozymes of it, 1a, 1b, and 1c, have been identified. Interestingly, the 1c isozyme was reported to show no enzymatic activity, but it was not clearly demonstrated whether this inactivity was due to its dysfunction or due to its poor expression. In the present study, we (a) expressed individual CPT1 isozymes in COS7 cells, (b) evaluated quantitatively their expression levels by Western blotting using the three bacterially expressed CPT1 isozymes as standards, and (c) evaluated their catalytic activities. With these experiments, we successfully demonstrated that the absence of the enzymatic activity of the 1c isozyme was due to its dysfunction. In addition, experiments on the preparation of standard CPT1 isozymes revealed that the 1c isozyme did not show the standard relationship between migration in an SDS-PAGE gel and molecular size. We further tried to determine why the 1c isozyme was inert by preparing chimeric CPT1 between 1a and 1c, but no clear conclusion could be drawn because one of the chimeric CPT1s was not sufficiently expressed.
(Keyword)
Acyl Coenzyme A / Animals / COS Cells / Carnitine O-Palmitoyltransferase / Cercopithecus aethiops / Electrophoresis, Polyacrylamide Gel / Gene Expression Regulation / Isoenzymes / Kinetics / Mitochondria, Liver
Naoto Okada, Kazuyoshi Kawazoe, Kazuhiko Teraoka, Toshihide Kujime, Masahiro Abe, Yasuo Shinohara and Kazuo Minakuchi : Identification of the Risk Factors Associated with Hypocalcemia Induced by Denosumab, Biological & Pharmaceutical Bulletin, Vol.36, No.10, 1622-1626, 2013.
(Summary)
Denosumab, a fully human monoclonal antibody that inhibits the receptor activator of nuclear factor-κB ligand, inhibits the activation of osteoclasts. Some clinical trials have shown that denosumab suppresses bone resorption in patients with advanced cancer, but hypocalcemia has been reported as a serious adverse effect after the administration of denosumab. It is difficult to predict hypocalcemia in such cases because the risk factors for denosumab-induced hypocalcemia have not been reported. Accordingly, the aim of the present study was to identify the risk factors for hypocalcemia induced by denosumab. We retrospectively reviewed the records of patients who had received denosumab at Tokushima University Hospital between April 2012 and May 2013. Fifty-three patients were analyzed and eleven patients had hypocalcemia after administration of denosumab. Univariate logistic regression analysis revealed that the patients who had not been administered zoledronic acid before receiving denosumab or had lower creatinine clearance (CCr) appeared to have a higher risk of hypocalcemia (p<0.05). The cut off value of CCr was 50.4 ml/min calculated by receiver-operator characteristics curves. Moreover, multivariate logistic regression analysis revealed that non-administration of zoledronic acid (odds ratio 10.43, p<0.05) and CCr less than 50.0 ml/min (odds ratio 5.90, p<0.05) were independent risk factors for denosumab-induced hypocalcemia. These findings provide useful information regarding the monitoring of hypocalcemia in patients receiving denosumab.
Takenori Yamamoto, Haruna Tamaki, Chie Katsuda, Kiwami Nakatani, Satsuki Terauchi, Hiroshi Terada and Yasuo Shinohara : Molecular basis of interactions between mitochondrial proteins and hydroxyapatite in the presence of Triton X-100, as revealed by proteomic and recombinant techniques., Journal of Chromatography. A, Vol.1301, 169-178, 2013.
(Summary)
Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100.
Mika Bandou, Xianqiong Zou, Yuka Hiroshima, Masatoshi Kataoka, Karen F. Ross, Yasuo Shinohara, Toshihiko Nagata, Mark C. Herzberg and Jun-ichi Kido : Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line, Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol.1829, No.9, 954-962, 2013.
(Summary)
S100A9 is a calcium-binding protein and subunit of antimicrobial calprotectin complex (S100A8/A9). Produced by neutrophils, monocytes/macrophages and keratinocytes, S100A9 expression increases in response to inflammation. For example, IL-1α produced by epithelial cells acts autonomously on the same cells to induce the expression of S100A8/A9 and cellular differentiation. Whereas it is well known that IL-1α and members of the IL-10 family of cytokines upregulate S100A8 and S100A9 in several cell lineages, the pathway and mechanism of IL-1α-dependent transcriptional control of S100A9 in epithelial cells are not established. Modeled using human epidermal keratinocytes (HaCaT cells), IL-1α stimulated the phosphorylation of p38 MAPK and induced S100A9 expression, which was blocked by IL-1 receptor antagonist, RNAi suppression of p38, or a p38 MAPK inhibitor. Transcription of S100A9 in HaCaT cells depended on nucleotides -94 to -53 in the upstream promoter region, based upon the use of deletion constructs and luciferase reporter activity. Within the responsive promoter region, IL-1α increased the binding activity of CCAAT/enhancer binding protein β (C/EBPβ). Mutated C/EBPβ binding sequences or C/EBPβ-specific siRNA inhibited the S100A9 transcriptional response. Hence, IL-1α is strongly suggested to increase S100A9 expression in a human epidermal keratinocyte cell line by signaling through the IL-1 receptor and p38 MAPK, increasing C/EBPβ-dependent transcriptional activity.
(Keyword)
Base Sequence / Calgranulin B / Cell Line / DNA Primers / Epidermis / Humans / Interleukin-1alpha / Keratinocytes / Polymerase Chain Reaction / RNA Interference / Transcription, Genetic / p38 Mitogen-Activated Protein Kinases
Kaori Abe, Y Hashimoto, S Yatsushiro, S Yamamura, Mika Bandou, Yuka Hiroshima, Jun-ichi Kido, M Tanaka, Yasuo Shinohara, Toshihiko Ooie, Yoshinobu Baba and Masatoshi Kataoka : Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip., PLoS ONE, Vol.8, No.1, e53620, 2013.
(Summary)
Sandwich enzyme-linked immunosorbant assay (ELISA) using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R(2) = 0.9994, TNF-α: R(2) = 0.9977), and the detection limit for each protein was 0.28 pg/ml and 0.46 pg/ml, respectively. Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis (IL-6: R(2) = 0.9954, TNF-α: R(2) = 0.9928). The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and reagents, and will be applicable to clinic diagnosis.
Shohei Yamamura, Shouki Yatsushiro, Yuka Nagasaki, Kaori Abe, Yasuo Shinohara and Masatoshi Kataoka : Detection of miRNA in cell cultures by using microchip electrophoresis with a fluorescence-labeled riboprobe., Sensors, Vol.12, No.6, 7576-7586, 2012.
(Summary)
The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe.
Kazuto Ohkura, Yuki Kawaguchi, Atsushi Tabata, Atsushi Yamamoto, Yasuo Shinohara, Hideaki Nagamune and Hitoshi Hori : Molecular Profiles of Cholesterol-dependent Cytolysin Family-derived 11mer Regions, Anticancer Research, Vol.32, No.6, 2343-2346, 2012.
(Summary)
Cholesterol-dependent cytolysins (CDCs) are secreted from various types of bacteria and are involved in various diseases (e.g. abscess formation). Traditional CDCs has a conserved 11mer region, which is a key structure in membrane recognition. Based on the X-ray data of traditional CDC perfringolysin O (PFO), molecular models of intermedilysin (ILY), pyolysin (PLO), vaginolysin (VLY), and Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) were constructed. The 11mer regions of these models were extracted, and their molecular features were analyzed. The dipole moments of these 11mer regions were classified into four types, and their stereo-hydrophobicity (dGW) was different. It was thought that these results influenced the species specificity and membrane recognition of each cytolysin. Traditional CDCs, ILY, PLO, and VLY consisted of four domains (domains 1 to 4). Domain 0 existed on the N-terminal side in Sm-hPAF in addition to these four domains. The 11mer sequence of Sm-hPAF is the same as that of VLY, but Sm-hPAF has slightly different characteristics (e.g. species specificity, membrane recognition, cholesterol dependency) compared to VLY. Dynamic structure analysis of domain 0 might clarify these differences.
Shohei Yamamura, Shouki Yatsushiro, Yuka Nagasaki, Kaori Abe, Yasuo Shinohara, Eiichi Tamiya, Yoshinobu Baba and Masatoshi Kataoka : Accurate detection of carcinoma cells by use of a cell microarray chip., PLoS ONE, Vol.7, No.3, e32370, 2012.
(Summary)
Accurate detection and analysis of circulating tumor cells plays an important role in the diagnosis and treatment of metastatic cancer treatment. A cell microarray chip was used to detect spiked carcinoma cells among leukocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene; and the formation of monolayers of leukocytes in the microchambers was observed. Cultured human T lymphoblastoid leukemia (CCRF-CEM) cells were used to examine the potential of the cell microarray chip for the detection of spiked carcinoma cells. A T lymphoblastoid leukemia suspension was dispersed on the chip surface, followed by 15 min standing to allow the leukocytes to settle down into the microchambers. Approximately 29 leukocytes were found in each microchamber when about 600,000 leukocytes in total were dispersed onto a cell microarray chip. Similarly, when leukocytes isolated from human whole blood were used, approximately 89 leukocytes entered each microchamber when about 1,800,000 leukocytes in total were placed onto the cell microarray chip. After washing the chip surface, PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD326 (EpCAM) monoclonal antibody solution were dispersed onto the chip surface and allowed to react for 15 min; and then a microarray scanner was employed to detect any fluorescence-positive cells within 20 min. In the experiments using spiked carcinoma cells (NCI-H1650, 0.01 to 0.0001%), accurate detection of carcinoma cells was achieved with PE-labeled anti-cytokeratin monoclonal antibody. Furthermore, verification of carcinoma cells in the microchambers was performed by double staining with the above monoclonal antibodies. The potential application of the cell microarray chip for the detection of CTCs was shown, thus demonstrating accurate detection by double staining for cytokeratin and EpCAM at the single carcinoma cell level.
Yuka Hiroshima, Mika Bandou, Yuji Inagaki, Chie Wada -Mihara, Masatoshi Kataoka, Hiromi Murata, Yasuo Shinohara, Toshihiko Nagata and Jun-ichi Kido : Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils, Journal of Periodontal Research, Vol.47, No.5, 554-562, 2012.
(Summary)
Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P-LPS). Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus-related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA(1c)) value was obtained from patients with diabetes mellitus-related periodontitis by a medical interview. Human neutrophils were cultured with P-LPS (0-1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P-LPS. The medium and cellular fractions were used for determination of resistin by ELISA. The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus-related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA(1c) value. The P-LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P-LPS-induced resistin release from neutrophils.
Takuya Hada, Yumiko Kato, Eriko Obana, Atsushi Yamamoto, Naoshi Yamazaki, Mitsuru Hashimoto, Takenori Yamamoto and Yasuo Shinohara : Comparison of two expression systems using COS7 cells and yeast cells for expression of heart/muscle-type carnitine palmitoyltransferase 1, Protein Expression and Purification, Vol.82, No.1, 192-196, 2012.
(Summary)
Carnitine palmitoyltransferase 1 (CPT1), catalyzing the transfer of the acyl group from acyl-CoA to carnitine to form acylcarnitine, is located at the outer mitochondrial membrane. Because it is easily inactivated by solubilization, expression systems using living cells are essential for its functional characterization. COS7 cells or yeast cells are often utilized for this purpose; however, the advantages/disadvantages of the use of these cells or the question as to how the CPT1 enzyme expressed by these cells differs are still uncertain. In this study, we characterized the heart/muscle-type isozyme of rat CPT1 (CPT1b) expressed by these two cellular expression systems. The mitochondrial fraction prepared from yeast cells expressing CPT1b showed 25% higher CPT1 activity than that obtained from COS7 cells. However, the expression level of CPT1b in the former was 3.8 times lower than that in the latter; and thus, under the present experimental conditions, the specific activity of CPT1b expressed in yeast cells was estimated to be approximately five times higher than that expressed in COS7 cells. Possible reasons for this difference are discussed.
Jun-ichi Kido, Mika Bandou, Yuka Hiroshima, Hiroyuki Iwasaka, Keisuke Yamada, Naoto Ohgami, Toshiyuki Namubu, Masatoshi Kataoka, Takenori Yamamoto, Yasuo Shinohara, Ikuko Sagawa and Toshihiko Nagata : Analysis of proteins in human gingival crevicular fluid by mass spectrometry, Journal of Periodontal Research, Vol.47, No.4, 488-499, 2012.
(Summary)
Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood-, cytoskeleton-, immunity-, inflammation- and lipid-related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S-transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1-antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.
Yusuke Ido, Takenori Yamamoto, Tatsuki Yoshitomi, Atsushi Yamamoto, Eriko Obana, Kazuto Ohkura and Yasuo Shinohara : Pseudogenes of rat VDAC1: 16 gene segments in the rat genome show structural similarities with the cDNA encoding rat VDAC1, with 8 slightly expressed in certain tissues., Mammalian Genome, Vol.23, No.3-4, 286-293, 2011.
(Summary)
BLAST analysis of the rat genome revealed the presence of 16 pseudogenes of isoform 1 of the mitochondrial voltage-dependent anion channel (VDAC1). Based on their structural characterization, it was concluded that these pseudogenes were formed by integration of VDAC1 cDNA into the genome, and subsequent rearrangements/mutations. By RT-PCR analysis using carefully designed primers that could not amplify the cDNA of genuine VDAC1, 8 of these 16 pseudogenes showed slight expression in certain tissues, but none of them seemed to encode a functional protein.
Eriko Obana, Takuya Hada, Takenori Yamamoto, Rei Kakuhata, Takuya Saze, Hirokazu Miyoshi, Tomoshige Hori and Yasuo Shinohara : Properties of signal intensities observed with individual probes of GeneChip Rat Gene 1.0 ST Array, an affymetric microarray system., Biotechnology Letters, Vol.34, No.2, 213-219, 2011.
(Summary)
For proper evaluation of the results of microarray experiments, it is important to understand how the signal intensities of individual probes are determined. Our previous studies revealed that signal intensities of individual probes in the Agilent array system (code G4131F) are largely dependent upon the location of the probes in the mRNA. In the present study, we examined the properties of signal intensities of individual probes in an Affymetrix array system (GeneChip Rat Gene 1.0 ST Array), in which a random primer fused to the T7 promoter sequence is employed. Distinct from the Agilent array system, individual probes used in this Affymetrix array system did not show the probe-location effects, but gave relatively diverse signal intensities. However, the diversities of the signal intensities of these individual probes were not due to experimental error.
Kazuto Ohkura, Yasuo Shinohara and Hitoshi Hori : Efficiency of Antimicrobial Defense: Molecular Flexibility of Natural Defensin and Artificial Bis-quaternary Ammonium Compound, Anticancer Research, Vol.31, No.7, 2561-2564, 2011.
(Summary)
Human α-defensins (such as HD5 and HD6) are typical bactericidal peptides. We examined the molecular features of HD5 and HD6 by molecular dynamics (MD) analysis. Molecular features of natural defensins and artificial bis-quaternary ammonium compounds (e.g. 1,6-polymethylenedithio)bis(1-octylpyridinium iodide: 4DTBP-m,8) were analyzed using molecular simulation techniques. HD5 and HD6 had different electrostatic potential profiles, which indicated the region-dependent hydrophobicity. 4DTBP-m,8 derivatives were significantly flexible, and many conformers existed. HD5 and HD6 indicated antimicrobial activity by restricted conformation.
Satoshi Kawashima, Takenori Yamamoto, Yuka Horiuchi, Kengo Fujiwara, Shunichi Gouda, Yuya Yoshimura, Atsushi Yamamoto, Yuki Inotani, Kikuji Yamashita, Seiichiro Kitamura, Hiroshi Terada, Makoto Kanematsu, Kozo Shishido and Yasuo Shinohara : S-15176 and its methylated derivative suppress the CsA-insensitive mitochondrial permeability transition and subsequent cytochrome c release induced by silver ion, and show weak protonophoric activity., Molecular and Cellular Biochemistry, Vol.358, No.1-2, 45-51, 2011.
(Summary)
A recent report has described that S-15176 (N-[(3,5-di-tert-butyl-4-hydroxy-1-thiophenyl)]-3-propyl-N'-(2,3,4-trimethoxybenzyl) piperazine), an anti-ischemic agent, inhibits the mitochondrial permeability transition (PT) induced by not only Ca(2+) and inorganic phosphate, but also by tert-butylhydroperoxide or phenylarsine oxide [Morin et al. (Biochem Pharmacol 72:911-918, 2006)]. In the present study, we tested the effects of S-15176 on the PT induced by Ag(+), PT of which is not suppressed by cyclosporin A or oligomycin. S-15176 was effective in suppressing the PT and the subsequent cytochrome c release induced by Ag(+), and hence, it was concluded to be a more universal PT inhibitor than cyclosporin A or oligomycin. In addition to the PT-suppression activity, S-15176 also showed weak protonophoric activity. Thus, we further tested to investigate whether the hydroxyl group of S-15176 was involved in its PT-suppression or weak protonophoric activities. The methylated derivative of S-15176 also showed both PT suppression and weak protonophoric activities; hence, the hydroxyl group of S-15176 was concluded not to be involved in these activities.
(Keyword)
Animals / Calcium / Cyclosporine / Cytochromes c / Dose-Response Relationship, Drug / Ions / Male / Methylation / Mitochondria, Liver / Mitochondrial Membrane Transport Proteins / Nephelometry and Turbidimetry / Oligomycins / Piperazines / Protons / Rats / Rats, Wistar / Silver
Masahiro Watanabe, Takenori Yamamoto, Atsushi Yamamoto, Eriko Obana, Kanami Niiyama, Takuya Hada, Toshihiko Ooie, Masatoshi Kataoka, Tomoshige Hori, Hitoshi Houchi and Yasuo Shinohara : Differential effects of cold exposure on gene expression profiles in white versus brown adipose tissue., Applied Biochemistry and Biotechnology, Vol.165, No.2, 538-547, 2011.
(Summary)
The thermogenic function of brown adipose tissue (BAT) is known to be markedly elevated when animals are exposed to the cold, and intensive studies have been carried out to understand the molecular basis enabling effective thermogenesis in cold-exposed animals. In this study, we used microarray analysis to examine the effects of cold exposure of animals on their gene expression profiles in white adipose tissue (WAT), which seems to function as a counterpart tissue of BAT. The results indicate that the effects of cold exposure on the gene expression profiles of WAT were much more moderate than the effects on those of BAT. Possible reasons for the different responses of BAT and WAT to cold exposure are discussed.
(Keyword)
brown adipose tissue / Adipose Tissue, White / Animals / Blotting, Northern / Cold Temperature / energy metabolism / Gene Expression Profiling / Gene Expression Regulation / Male / Mitochondrial Proteins / Oligonucleotide Array Sequence Analysis / Rats / Thermogenesis
We describe the potential of microchip electrophoresis with a Hitachi SV1210, which can be used to evaluate the integrity of total RNA, for the analysis of mRNA expression. The ribonuclease (RNase) protection assay was performed by using microchip electrophoresis with cyanine 5 (Cy5) labeled 248-base antisense RNA probe (riboprobe) encoding adipose-type fatty acid binding protein (A-FABP) as the riboprobe. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary strand RNA. Results were obtained in 120 s, and the same amount of Cy5-labeled antisense riboprobe as used in the conventional method can be used. Furthermore, 8 times more sensitive detection of mRNA by microchip electrophoresis could be obtained. An obvious increase in the mRNA expression of A-FABP, which is known as a differentiation marker of adipocytes, occurred during the adipocyte differentiation of 3T3-L1 cells. These results clearly indicate the potential of microchip electrophoresis for the analysis of mRNA expression in cells.
Naoto Okada, Takenori Yamamoto, Masahiro Watanabe, Yuuya Yoshimura, Eriko Obana, Naoshi Yamazaki, Kazuyoshi Kawazoe, Yasuo Shinohara and Kazuo Minakuchi : Identification of TMEM45B as a protein clearly showing thermal aggregation in SDS-PAGE gels and dissection of its amino acid sequence responsible for this aggregation., Protein Expression and Purification, Vol.77, No.1, 118-123, 2011.
(Summary)
SDS-PAGE is one of the most powerful experimental techniques used for the separation of proteins, and most proteins are separated according to their molecular size by this technique. However, exceptional proteins showing abnormal behavior in SDS-PAGE gels are known to exist. Thermal aggregation, rarely observed with membrane proteins, is one of the exceptional behaviors of proteins during SDS-PAGE, but detailed characterization of this aggregation has not yet been achieved. In the present study, we found that a putative membrane protein, TMEM45B, very clearly showed properties of thermal aggregation when it was expressed in COS7 cells and subjected to SDS-PAGE. We dissected the region of TMEM45B responsible for this aggregation, and found that of the seven putative transmembrane domains, a region comprising the 4th to 7th ones was essential for the thermal aggregation properties. We also demonstrated that these transmembrane domains, 4th to 7th, of TMEM45B conferred thermal aggregation properties on other proteins, by fusing this amino acid sequence to target proteins. The molecular mechanism causing thermal aggregation by TMEM45B is still uncertain, but TMEM45B could be utilized as a nice model to show clear thermal aggregation in SDS-PAGE gels.
Takenori Yamamoto, Atsushi Yamamoto, Masahiro Watanabe, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Quantitative evaluation of the effects of cold exposure of rats on the expression levels of ten FABP isoforms in brown adipose tissue., Biotechnology Letters, Vol.33, No.2, 237-242, 2010.
(Summary)
We quantitatively examined the transcript levels of ten fatty acid-binding protein (FABP) isoforms in the brown adipose tissue (BAT) of rats kept at room temperature and of rats exposed to the cold by Northern blotting using the synthesized RNA of each isoform as an external standard. FABP3-5 were expressed in BAT of both rats maintained at room temperature and those exposed to the cold. FABP4 was the most abundantly expressed isoform, but its transcript level was not significantly affected by cold exposure. FABP3 was slightly expressed in the BAT of rats maintained at room temperature and its transcript level was elevated ten fold by cold exposure. FABP5 was also elevated four fold by cold exposure but the amount of its mRNA in BAT was negligible.
(Keyword)
Adipose Tissue, Brown / Animals / Blotting, Northern / Cold Temperature / Fatty Acid-Binding Proteins / Gene Expression Profiling / Protein Isoforms / RNA, Messenger / Rats
Kazuto Ohkura, Katsumi Fukino, Yasuo Shinohara and Hitoshi Hori : N-Acetyl Transferase 2 Polymorphisms Associated with Isoniazid Pharmacodynamics: Molecular Features for Ligand Interaction, Anticancer Research, Vol.30, No.8, 3177-3180, 2010.
(Summary)
Isoniazid is mainly metabolized by arylamine N-acetyltransferase 2 (NAT2). Rapid acetylator types have NAT2*4/*4 alleles. Intermediate acetylator types have any of the following alleles: NAT2*4/*5, *4/*6, or *4/*7. Slow acetylator types do not have the NAT2*4 allele. We examined molecular features of these NAT2 molecules. Structures of NAT2*5, *6, and *7 were constructed based on X-ray data of human NAT2*4 using a molecular modeling technique. The NAT2*4 molecule mostly occupied a positive electrostatic potential field. Ile(114) and Arg(197), which are mutation sites of NAT2*4 to NAT2*5 and NAT2*6, were located in the peripheral part of the positive field. Gly(286), the mutation site from NAT2*4 to NAT2*7, was located near coenzyme A (CoA) in the boundary of the positive and negative fields. Nonbinding energies between NAT2s and isoniazid were larger than those of CoA. Molecular polymorphism appears to influence the reactivity between NAT2 and the external ligand.
Chie Katsuda, Kanami Niiyama, Eriko Obana, Takenori Yamamoto, Yumiko Katou, Masatoshi Kataoka, Kazuto Ohkura and Yasuo Shinohara : Specific formation of trypsin-resistant micelles on a hydrophobic peptide observed with Triton X-100 but not with octylglucoside., Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1798, No.11, 2090-2093, 2010.
(Summary)
The manner of interaction of the coat peptide of the Pf3 phage (Pf3 peptide) with lipid bilayers has been extensively studied. Presently, we designed a derivative of the Pf3 peptide, referred to as the DDRK peptide, and subjected it to trypsin digestion to understand its physicochemical properties. In the presence of Triton X-100 used for solubilization of the peptide, digestion of DDRK with trypsin caused specific cleavage at the lysine (Lys) residue in its N-terminal region but not at other Lys residues or at the arginine residue. As the N-terminal region of the DDRK peptide is relatively hydrophilic, but its remaining region is hydrophobic, this hydrophobic region of the peptide would be expected to be coated by Triton micelles. Thus, we propose that the presence of such micelles protected against cleavage there, leading to selective cleavage by trypsin of the DDRK peptide at its hydrophilic Lys residue in the N-terminal part of the molecule. However, such a protective effect on the DDRK peptide against trypsin digestion was not observed with octylglucoside. The observed results are important for better understanding of the manner of interaction between detergents and hydrophobic peptides.
Yuka Hiroshima, Mika Bandou, Masatoshi Kataoka, Yasuo Shinohara, MC Herzberg, KF Ross, Yuji Inagaki, Toshihiko Nagata and Jun-ichi Kido : Shosaikoto increases calprotectin expression in human oral epithelial cells., Journal of Periodontal Research, Vol.45, No.1, 79-86, 2010.
(Summary)
BACKGROUND AND OBJECTIVE: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1alpha (IL-1alpha). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1alpha in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1alpha in oral epithelial cells. MATERIAL AND METHODS: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 microg/mL) in the presence or absence of anti-IL-1alpha or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1alpha secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1alpha was analyzed by RT-PCR and by quantitative real-time PCR. RESULTS: Shosaikoto (25 microg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1alpha-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1alpha or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. CONCLUSION: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1alpha.
Takenori Yamamoto, Mizuki Ohashi, Sho Mizutani, Yuuya Yoshimura, Eriko Obana, Hiroshi Terada and Yasuo Shinohara : Use of Highly Purified and Mixed Antibodies for Simultaneous Detection of Multiple Protein Species Released from Mitochondria upon Induction of the Permeability Transition., Applied Biochemistry and Biotechnology, 2010.
(Summary)
Concomitant with the induction of the mitochondrial permeability transition (PT), cytochrome c is released from mitochondria into the cytosol where it triggers subsequent steps of cellular apoptosis. Thus, inducers of the mitochondrial PT would become "seed compounds" of regulators of apoptosis. However, when we examine the actions of certain chemicals on the release of mitochondrial cytochrome c, the behaviors of not only cytochrome c but also multiple mitochondrial protein species must be carefully examined because the mitochondrial PT and release of proteins from mitochondria occur in diverse manners. In the present study, we examined whether it is possible to measure the behaviors of multiple protein species in a single experiment using purified and mixed antibodies. The results obtained clearly indicate that this procedure would be applicable for high-throughput screening of regulators of apoptosis. Further requirements necessary for the establishment of a useful screening system for apoptosis regulators are discussed.
Yoshihiro Umemoto, Masatoshi Kataoka, Shouki Yatsushiro, Shouhei Yamamura, Toshihiko Ooie, Jun-ichi Kido, Takenori Yamamoto, Yasuo Shinohara and Yoshinobu Baba : Analysis of DNA ligation by microchip electrophoresis., Journal of Pharmaceutical and Biomedical Analysis, Vol.52, No.2, 323-328, 2010.
(Summary)
We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to determine DNA sizes between 500 and 5000 bp with good quantification (DNA concentration, <8 ng/l) within 5 min, for the analysis of DNA ligation. On analysis of an electropherogram of a ligation mixture of the pTAC1-T vector and a 789 bp PCR-amplified DNA fragment, the presence of recombinant DNA was easily detected by comparison with an electropherogram obtained without ligase. On analysis of a ligation mixture of pUC19/Eco RI without alkaline phosphatase treatment and a 667 bp Eco RI-digested fragment of foreign DNA, several peaks observed in the electropherogram corresponded to the formation of monomeric and polymeric insert DNAs, self-ligated vector DNA, and recombinant DNA. On the other hand, several peaks were also observed in the electropherogram of the ligation mixture of pUC19/Eco RI with alkaline phosphatase treatment and the 667 bp Eco RI-digested fragment of foreign DNA, the fluorescence intensity corresponding to recombinant DNA apparently being increased. These results indicate the potential of microchip electrophoresis for the analysis of DNA ligation, it offering high resolution in a short time.
Taisuke Matsuo, Atsushi Yamamoto, Takenori Yamamoto, Kaoru Otsuki, Naoshi Yamazaki, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Replacement of C305 in heart/muscle-type isozyme of human carnitine palmitoyltransferase I with aspartic acid and other amino acids., Biochemical Genetics, Vol.48, No.3-4, 193-201, 2010.
(Summary)
Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.
Mika Bandou, Yuka Hiroshima, Masatoshi Kataoka, MC Herzberg, KF Ross, Yasuo Shinohara, Takenori Yamamoto, Toshihiko Nagata and Jun-ichi Kido : Modulation of calprotectin in human keratinocytes by keratinocyte growth factor and interleukin-1alpha., Immunology and Cell Biology, Vol.88, No.3, 328-333, 2010.
(Summary)
Calprotectin is an antimicrobial complex composed of the S100A8 and S100A9 protein family subunits. Contributing to innate immunity, calprotectin expression is increased by interleukin-1alpha (IL-1alpha), which modulates keratinocyte differentiation. Keratinocyte growth factor (KGF) is produced by mesenchymal cells and has a mitogenic activity for epithelial cells. In this study, we investigated the effect of KGF on calprotectin expression in keratinocytes and modulation by IL-1alpha. Human keratinocytes were cultured with KGF in the presence or absence of a KGF receptor (KGFR) inhibitor or mitogen-activated protein kinase (MAPK) inhibitors. Calprotectin (S100A8/S100A9) expression was determined by northern blotting and enzyme-linked immunosorbent assay, respectively, whereas MAPK phosphorylation was analyzed by western blot analysis. KGF significantly decreased the expression of S100A8/S100A9-specific mRNAs and calprotectin protein. In the presence of KGF, KGFR inhibitor or extracellular-regulated kinase inhibitor restored KGF-downregulated expression of S100A8/S100A9. KGF increased IL-1alpha expression in keratinocytes, whereas IL-1alpha increased KGF expression in fibroblasts. Cocultured fibroblast and keratinocytes showed lower S100A8/S100A9 mRNA expression than keratinocytes alone in the presence or absence of IL-1alpha or KGF. These results suggest that fibroblast-derived KGF reduces or restricts calprotectin expression in keratinocytes, which supports our hypothesis that calprotectin expression in keratinocytes is modulated by factors associated with epithelial-mesenchymal interactions.
Shouki Yatsushiro, Shohei Yamamura, Yuka Nagasaki, Yasuo Shinohara, Eiichi Tamiya, Toshihiro Horii, Yoshinobu Baba and Masatoshi Kataoka : Rapid and highly sensitive detection of malaria-infected erythrocytes using a cell microarray chip., PLoS ONE, Vol.5, No.10, 2010.
(Summary)
The potential of the cell microarray chip for the detection of malaria-infected erythrocytes was shown, it offering 10-100 times higher sensitivity than that of conventional light microscopy and easy operation in 15 min with purified erythrocytes.
Kazuto Ohkura, Hitoshi Hori and Yasuo Shinohara : Role of C-terminal region of yeast ADP/ATP carrier 2 protein: dynamics of flexible C-terminal arm., Anticancer Research, Vol.29, No.11, 4897-4900, 2009.
(Summary)
The ADP/ATP carrier catalyzes the exchange of ADP and ATP across the inner mitochondrial membrane. The molecular dynamics of modeled yeast type 2 AAC (yAAC2) was analyzed and molecular parameters were determined. The yAAC2 C-terminal moved flexibly and a negative electrostatic potential field (ESP) was located in the C-terminal region. The ESP field is always located in the C-terminal area during C-terminal truncation (d1-d9). Further C-terminal truncation occurred on field invagination into the core region (d11, d14, d16). The 2-6 C-terminal amino acid truncation did not affect the biological activity. The d7-d9 truncated mutants lost their biological function. A critical point in yAAC2 function was shown between d6 and d7 C-terminal truncation. The C-terminal structure of yAAC2 is thought to be involved in biological function control.
Akiko Yamada, Takenori Yamamoto, Yuya Yoshimura, Shunichi Gouda, Satoshi Kawashima, Naoshi Yamazaki, Kikuji Yamashita, Masatoshi Kataoka, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara : Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions., Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1787, No.12, 1486-1491, 2009.
(Summary)
Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca(2+) within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca(2+) has remained obscure. In this study, we examined in detail the effects of Ca(2+) on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca(2+) under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca(2+) were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca(2+) was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca(2+), the release of cytochrome c from mitochondria was also observed.
(Keyword)
calcium / Cytochromes c / Mitochondrial Membrane Transport Proteins / Yeasts
Takenori Yamamoto, Atsushi Yamamoto, Masahiro Watanabe, Taisuke Matsuo, Naoshi Yamazaki, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Classification of FABP isoforms and tissues based on quantitative evaluation of transcript levels of these isoforms in various rat tissues., Biotechnology Letters, Vol.31, No.11, 1695-1701, 2009.
(Summary)
In mammals, 10 isoforms of fatty acid-binding protein (FABP) are expressed in various tissues. To understand the role of multiple FABP isoforms, we have quantitatively examined the transcript levels of individual FABP isoforms in each of various tissues by Northern blotting using synthesized RNAs corresponding to the mRNA of each isoform as external standards. As a result, absolute transcript levels of individual FABP isoforms expressed in each tissue were successfully determined. The 10 FABP isoforms were classified into three categories: (1) isoforms FABP7 and 12 were not markedly expressed in any tissue examined; (2) isoforms showing certain transcript levels in multiple tissues; and (3) isoforms FABP6, 8, and 9, expressed at certain levels in one particular tissue. Based on the expression profiles of the isoforms, individual tissues were also classified into three groups: (1) tissues in which high-level expression of FABP isoforms was not observed, (2) tissues in which multiple FABP isoforms were expressed at certain levels, and (3) tissues in which a single FABP isoform was dominantly expressed at a certain level. These results give a better understanding of the meaning of the presence of multiple FABP isoforms in mammals.
(Keyword)
Animals / DNA Probes / DNA, Complementary / Fatty Acid-Binding Proteins / Gene Expression Profiling / Gene Expression Regulation / Organ Specificity / Protein Isoforms / RNA, Messenger / Rats
Kazuto Ohkura, Yuki Kawguchi, Yasuo Watanabe, Yasuhiro Masubuchi, Yasuo Shinohara and Hitoshi Hori : Flexible Structure of Cytochrome P450: Promiscuity of Ligand Binding in the CYP3A4 Heme Pocket, Anticancer Research, Vol.29, No.3, 935-942, 2009.
(Summary)
CYP3A4 is the most abundant xenobiotic-metabolizing cytochrome P450 isoform. We examined the structural features of the CYP3A4 molecule with regard to ligand access. The deleted amino acid sequences of X-ray data sets of CYP3A4s were complemented by molecular modeling techniques. Molecular features of the ligand accessible regions in CYP3A4 were analyzed and their molecular parameters (e.g. dipole moment, solvation free energy, electrostatic potential fields) were determined. Three ligand accessible regions (region 1-3) were present in erythromycin-bound CYP3A4, and these dipole moments indicated the same features as ketoconazole- or metyrapone-bound CYP3A4 molecules. In progesterone-bound CYP3A4, four candidate ligand accessible regions were observed and progesterone could be bound by two selected ligand accessible regions. The heme pocket of CYP3A4 is very flexible and is able to interact with various types of substrate.
Akiko Yamada, Takenori Yamamoto, Naoshi Yamazaki, Kikuji Yamashita, Masatoshi Kataoka, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara : Differential permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes as revealed by proteomics analysis of proteins released from mitochondria., Molecular & Cellular Proteomics, Vol.8, No.6, 1265-1277, 2009.
(Summary)
It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224-5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed.
Yoshihiro Umemoto, Masatoshi Kataoka, Shouki Yatsushiro, Masahiro Watanabe, Jun-ichi Kido, Rei Kakuhata, Takenori Yamamoto, Yasuo Shinohara and Yoshinobu Baba : Sequential analysis of RNA synthesis by microchip electrophoresis., Analytical Biochemistry: Methods in the Biological Sciences, Vol.388, No.1, 161-163, 2009.
(Summary)
We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to evaluate the integrity of total RNA, for the analysis of synthesized RNA. There was little interference by DNA and/or the components of the in vitro transcription system with the microchip electrophoresis. The fluorescence intensity corresponding to the synthesized RNA increased in a time-dependent manner as to the RNA synthesis reaction on sequential analysis. A result can be obtained in 160 s and only 1/10 aliquots of samples, compared with the conventional method, are required. These results indicate the potential of microchip electrophoresis for sequential analysis of RNA synthesis.
Taisuke Matsuo, Eriko Obana, Takenori Yamamoto, Rei Kakuhata, Naoshi Yamazaki, Masatoshi Kataoka, Yoshinobu Baba, Tomoshige Hori and Yasuo Shinohara : Construction of plasmids suitable for in vitro synthesis of full-length mRNAs having a 3'-poly(A)+tail., Biotechnology Letters, Vol.31, No.2, 203-207, 2009.
(Summary)
In vitro synthesized mRNAs are often used as calibrants in gene expression analysis. In the case of an analytical procedure for gene expression containing a process of reverse transcription such as microarray analysis, full-length mRNAs having a 3'-poly(A)+tail are required as calibrants. However, the preparation of full-length mRNAs having a 3'-poly(A)+tail by using ordinary plasmid vectors is difficult. In this study, we established two plasmid vectors in which the nucleotide sequence corresponding to the 3'-poly(A)+tail were included. By simply inserting full-length cDNAs lacking their 3'-poly(A)+tail into established plasmid vectors, full-length mRNAs having a 3'-poly(A)+tail were successfully prepared.
Taisuke Matsuo, Takenori Yamamoto, Chie Katsuda, Kanami Niiyama, Atsushi Yamamoto, Naoshi Yamazaki, Kazuto Ohkura, Masatoshi Kataoka and Yasuo Shinohara : Substitution of certain amino acids in a short peptide causes a significant difference in their immunoreactivities with antibodies against different epitopes: evidence for possible folding of the peptide on a nitrocellulose or PVDF membrane., Biologicals, Vol.37, No.1, 44-47, 2009.
(Summary)
Substitution of amino acids in a peptide caused remarkable differences in its immunoreactivities with antibodies against 3 epitopes in the immobilized peptide. The observed differences in immunoreactivities among the peptides were not due to the differences in efficiencies of their transfer onto nitrocellulose or PVDF membranes. Rather, possible folding of the peptide on the membrane was considered to be the reason for their distinct immunoreactivities with the antibodies.
Akihiro Iwahashi, Aoi Ishii, Naoshi Yamazaki, Mutsuru Hashimoto, Kazuto Okura, Masatoshi Kataoka, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier., Mitochondrion, Vol.8, No.2, 196-204, 2008.
(Summary)
Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed.
Naoshi Yamazaki, Taisuke Matsuo, Miho Kurata, Makiko Suzuki, Takehisa Fujiwaki, Seiji Yamaguchi, Hiroshi Terada and Yasuo Shinohara : Substitutions of Three Amino Acids in Human Heart/Muscle Type Carnitine Palmitoyltransferase I Caused by Single Nucleotide Polymorphisms, Biochemical Genetics, Vol.46, No.1-2, 54-63, 2008.
(Summary)
Heart/muscle type carnitine palmitoyltransferase I (M-CPTI) catalyzes the rate-limiting step of mitochondrial long-chain fatty acid (LCFA) oxidation in muscle and adipose tissue. Three replacements of nucleotides resulting in missense mutations of I66V, S427C, and E531K were observed in the M-CPTI gene of patients showing abnormal fatty acid metabolism. These nucleotide replacements were found to be common single nucleotide polymorphisms (SNPs) of this gene and not specific to patients. The question of whether these missense mutations caused by SNPs alter the functional properties of M-CPTI remains unanswered. Thus, we examined whether these missense mutations are associated with any changes in the enzymatic properties of M-CPTI. None of these mutations was found to cause remarkable alteration of its enzymatic properties. Based on the comparison of amino acid sequences of M-CPTI among different animal species, the roles of these amino acids in the enzyme are discussed.
Rei Kakuhata, Masahiro Watanabe, Takenori Yamamoto, Eriko Obana, Naoshi Yamazaki, Masatoshi Kataoka, Toshihiko Ooie, Yoshinobu Baba, Tomoshige Hori and Yasuo Shinohara : Importance of probe location for quantitative comparison of signal intensities among genes in microarray analysis., Journal of Biochemical and Biophysical Methods, Vol.70, No.6, 926-931, 2008.
(Summary)
In our previous studies, we demonstrated that expression levels of genes determined by Agilent oligoarray system, code G4130A, could be quantitatively evaluated by spike-in of synthetic full-length mRNAs as standards [Kakuhata R, Watanabe M, Yamamoto T, Akamine R, Yamazaki N, Kataoka M, et al. Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis, J Biochem Biophys Methods 2007;70:755-60]. However, in its successor version (Agilent oligo array system, code G4131F), which was established to enable gene expression analysis over a much wider dynamic range, multiple probes are often utilized for evaluation of expression levels of individual genes; and they showed markedly distinct signal intensities. This result indicates that the observed signal intensities in this new version seemed not to simply reflect the transcript levels of individual genes. To understand the factors influencing the signal intensities of probes, we characterized the properties of the probes used in this new array system and the cRNAs formed during the labeling process. Analysis of cRNAs in the reaction mixture, which were hybridized with the arrays, revealed that the cRNAs were not fully transcribed under the conditions used. For this reason, probes located at the 5' side of the message were found to give lower signals than those at the 3' end; and the observed signal intensities were dependent upon the location of probes in the mRNA. Analysis of the correlation between signal intensities and locations on mRNAs for larger numbers of probes also supported the idea that probe location is one of the major determinants of signal intensities of probes in microarray analysis.
Masahiro Watanabe, Takenori Yamamoto, Rei Kakuhata, Naoto Okada, Kazuaki Kajimoto, Naoshi Yamazaki, Masatoshi Kataoka, Yoshinobu Baba, Toshiaki Tamaki and Yasuo Shinohara : Synchronized changes in transcript levels of genes activating cold exposure-induced thermogenesis in brown adipose tissue of experimental animals., Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1777, No.1, 104-112, 2008.
(Summary)
To identify genes whose expression in brown adipose tissue (BAT) is up- or down-regulated in cold-exposed rats, we performed microarray analysis of RNA samples prepared from the BAT of cold-exposed rats and of rats kept at room temperature. Previously reported elevations of transcript levels of uncoupling protein (UCP1), type II iodothyronine deiodinase (DIO2), and type III adenylate cyclase (AC3) in the BAT of cold-exposed rats over those in that of rats maintained at room temperature were confirmed. In addition to these changes, remarkable elevations of the transcript levels of several genes that seemed to be associated with the processes of cell-cycle regulation and DNA replication were detected in the BAT of cold-exposed rats, possibly reflecting the significant proliferation of brown adipocytes in response to cold exposure. Up-regulation of the gene encoding sarcomeric mitochondrial type creatine kinase in the BAT of cold-exposed rats was also detected by microarray analysis, but subsequent Northern analysis revealed that the expression of not only the sarcomeric mitochondrial type enzyme, but also that of 2 other subtypes, i.e., cytoplasmic brain type and cytoplasmic muscle type, was elevated in the BAT of cold-exposed rats. Microarray analysis also revealed a significant expression of myoglobin in BAT and its elevation in the BAT of cold-exposed rats, and this result was supported by calibrated Northern analysis. On the contrary, several genes such as regulator of G-protein signaling 2 and IMP dehydrogenase 1 were down-regulated in the BAT of cold-exposed rats. The physiological meaning of these changes accompanying cold exposure was discussed.
Yuma Yamada, Hidetaka Akita, Hiroyuki Kamiya, Kentaro Kogure, Takenori Yamamoto, Yasuo Shinohara, Kikuji Yamashita, Hideo Kobayashi, Hiroshi Kikuchi and Hideyoshi Harashima : MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion., Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1778, No.2, 423-432, 2007.
(Summary)
Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.
Mami Hino, Masatoshi Kataoka, Kazuaki Kajimoto, Takenori Yamamoto, Jun-ichi Kido, Yasuo Shinohara and Yoshinobu Baba : Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes., Journal of Biotechnology, Vol.133, No.2, 183-189, 2007.
(Summary)
The efficiency of protein synthesis for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was examined with several in vitro coupled transcription/translation protein synthesis systems based on Escherichia coli lysate, wheat germ, or reticulocyte lysate, and an in vitro translation system based on wheat germ extract. A significant amount of protein synthesis was observed only in systems based on E. coli using pET/G3PDH as the expression vector. A remarkable increase of protein synthesis was obtained in wheat germ using a pT(N)T expression vector which contains a 5'-globin leader sequence and a synthetic poly(A)(30) tail instead of pET. A significant difference of T7 RNA polymerase presence by Western blot analysis was not observed in the first four systems, and the difference of total RNA presence in each reaction mixture by Northern blot analysis seemed unrelated to protein synthesis. Although a small amount of protein was synthesized using RNA-encoding G3PDH transcribed in vitro with pET/G3PDH by an in vitro translation system, an extreme increase was observed using transcribed RNA with pEU/G3PDH, which contains T7 RNA promoter and a translation enhancer, Omega sequence. These results suggest that the presence of an enhancer sequence for translation is one of the critical steps for protein synthesis by a eukaryotic cell-free protein synthesis system.
Mika Bandou, Yuka Hiroshima, Masatoshi Kataoka, Yasuo Shinohara, MC Herzberg, KF Ross, Toshihiko Nagata and Jun-ichi Kido : Interleukin-1alpha regulates antimicrobial peptide expression in human keratinocytes., Immunology and Cell Biology, Vol.85, No.7, 532-537, 2007.
(Summary)
Human epidermis and epithelium serve as physiologic barriers to protect against noxious and infectious agents. Contributing to the defense against infection, epithelial cells express antimicrobial peptides (AMPs). The expression of AMPs in keratinocytes is generally regulated directly by bacteria and indirectly by proinflammatory cytokines. Bacteria may also regulate AMP expression by inducing keratinocyte expression of the autonomous proinflammatory cytokine, interleukin-1alpha (IL-1alpha). To test the hypothesis that AMP expression may be regulated by cell autonomous cytokines, we investigated the effect of IL-1alpha on the expression of AMPs in human keratinocytes (HaCaT cells) by microarray, northern blot, reverse transcriptase (RT)-PCR and western blot analyses. IL-1alpha increased expression of mRNA in a dose- and time-dependent manner specific for lipocalin 2, S100A8, S100A9 and secretory leukocyte protease inhibitor (SLPI) more than twofold relative to nonstimulated cells (control), and slightly upregulated S100A7 and beta-defensin-2. Furthermore, the expression of lipocalin 2, S100A7, S100A8, S100A9 and SLPI proteins were upregulated by IL-1alpha. On the other hand, HaCaT cells expressed mRNA specific for other AMPs, including cystatin 3, adrenomedullin, RNase-7 and mucin 5, which were unaffected by IL-1alpha treatment. These results suggest that the autonomous keratinocyte cytokine, IL-1alpha, selectively upregulates the expression of AMPs which may modulate innate epithelial cell immunity in skin and mucosa.
Rei Kakuhata, Masahiro Watanabe, Takenori Yamamoto, Rie Akamine, Naoshi Yamazaki, Masatoshi Kataoka, Satoshi Fukuoka, Mitsuru Ishikawa, Toshihiko Ooie, Yoshinobu Baba, Tomoshige Hori and Yasuo Shinohara : Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis., Journal of Biochemical and Biophysical Methods, Vol.70, No.5, 755-760, 2007.
(Summary)
To examine the possible usefulness of in vitro synthesized RNA as standards in microarray analysis, we prepared full-length mRNAs encoded by 3 rat metabolic genes for heart/muscle type carnitine palmitoyltransferase I (M-CPTI), uncoupling protein (UCP1), and heart/muscle type fatty acid-binding protein (H-FABP). Artificial RNA samples were prepared by adding known amounts of these synthetic mRNAs to total RNA from rat liver, and transcript levels of various genes were compared between the prepared artificial RNA samples and total RNA samples of rat liver by using an Agilent oligo microarray system. Upon the addition of these synthetic RNAs, signals from the DNA spots corresponding to these 3 genes were elevated, but those from the DNA spots representing other genes were not markedly influenced. Using the ratio of the increase in signal intensity of DNA spot to the amount of added RNA, we estimated the expression levels of several genes and compared them with the absolute expression levels determined by calibrated Northern analysis. As a result, the absolute transcript levels of 3 genes encoding acidic ribosomal phosphoprotein P0, type-1 voltage-dependent anion channel (VDAC1), and type-2 glucose transporter (GLUT2) were successfully estimated by this procedure. Furthermore, genes specifically expressed in certain tissues such as UCP1 were concluded to be good candidates as standards for use in microarray analysis.
Rie Akamine, Takenori Yamamoto, Masahiro Watanabe, Naoshi Yamazaki, Masatoshi Kataoka, Mitsuru Ishikawa, Toshihiko Ooie, Yoshinobu Baba and Yasuo Shinohara : Usefulness of the 5' region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice, and humans as a standard probe for gene expression analysis in different tissues and animal species., Journal of Biochemical and Biophysical Methods, Vol.70, No.3, 481-486, 2007.
(Summary)
Housekeeping genes are often used as internal standards for gene expression analysis. When steady-state transcript levels of 4 typically used housekeeping genes, i.e., beta-actin, glyceraldehyde 3-phosphate dehydrogenase, cyclophilin, and acidic ribosomal phosphoprotein P0 (36B4), were evaluated in various rat tissues, the 36B4 gene seemed to be the most suitable as a standard to compare the expression levels of genes among different tissues. Next, for possible quantitative comparison of the expression level of this gene among different animal species, we compared the nucleotide sequence of the cDNA of 36B4 among rats, mice, and humans. As a result, highly conserved regions showing more than 97.5% identities were observed in the 5' portion of its open reading frame. When samples of synthesized mRNA encoding rat, mouse, and human 36B4 were hybridized with the entire cDNA encoding rat 36B4 as a probe, hybridization signals of mRNAs of mouse and human 36B4 were much weaker than those of mRNA encoding rat 36B4. However, when they were hybridized with an oligonucleotide probe corresponding to the highly conserved regions, they showed similar signal intensities. Thus, these highly conserved regions of the cDNA encoding 36B4 were concluded to be an effective standard for use in gene expression analysis.
Noriko Hayashi, Jun-ichi Kido, (名) Suryono, Reiko Kido, Chie Wada -Mihara, Masatoshi Kataoka, Yasuo Shinohara and Toshihiko Nagata : Regulation of Calprotectin Expression by IL-1α and TGF-β in Human Gingival Keratinocytes, Journal of Periodontal Research, Vol.42, No.1, 1-7, 2007.
(Summary)
Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation. Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay. The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta. The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.
Takenori Yamamoto, Akiko Yamada, Masahiro Watanabe, Yuya Yoshimura, Naoshi Yamazaki, Yoshiyuki Yoshimura, Takashi Yamauchi, Masatoshi Kataoka, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara : VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues., Journal of Proteome Research, Vol.5, No.12, 3336-3344, 2006.
(Summary)
The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.
(Keyword)
Amino Acid Sequence / Animals / Base Sequence / Chromatography, Liquid / DNA Primers / Electrophoresis, Gel, Two-Dimensional / Electrophoresis, Polyacrylamide Gel / Immunoblotting / Mass Spectrometry / Mitochondrial Proteins / Molecular Sequence Data / Protein Isoforms / Rats / Sequence Analysis, DNA / Voltage-Dependent Anion Channel 1
Akihiro Iwahashi, Yoshitaka Kihira, Eiji Majima, Hiroshi Terada, Naoshi Yamazaki, Masatoshi Kataoka and Yasuo Shinohara : The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state, Mitochondrion, Vol.6, No.5, 245-251, 2006.
(Summary)
To detect structural changes in the second cytosolic loop of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae AAC2, we prepared 20 single cysteine mutants by replacing each amino acid in the S213 to L232 region. All single cysteine mutants were fully functional, because they could restore growth on glycerol of a yeast strain lacking functional ADP/ATP carriers. First, these single-Cys mutants were treated with carboxyatractyloside to lock the carrier in the cytosolic state or with bongkrekic acid to generate the matrix state, and then with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) to probe accessibility. The amino acid residues S213C, L214C, F231C and L232C were not labeled, indicating that these 4 residues must have been buried in the membrane, whereas the region between residues K215 and S230 is accessible to labeling and must, therefore, have protruded into the aqueous phase. Residue L218C showed strong resistance against EMA labeling regardless of the state of the carrier, but the reason for such behavior is unclear. On the contrary, the labeling of the residues between F227C and S230C was strongly dependent on the state of the carrier. Thus, the C-terminal region of the second cytosolic loop in AAC2 changes its environment when the carrier cycles between the matrix and cytosolic state.
(名) Suryono, Jun-ichi Kido, Noriko Hayashi, Masatoshi Kataoka, Yasuo Shinohara and Toshihiko Nagata : Norepinephrine Stimulates Calprotectin Expression in Human Monocytic Cells, Journal of Periodontal Research, Vol.41, No.3, 159-164, 2006.
(Summary)
Calprotectin is composed of two proteins, S100A8 and S100A9, which are S100 family members, and is detected in gingival crevicular fluid and gingival tissue with inflammation. The release and production of calprotectin are regulated by lipopolysaccharides of periodontopathic bacteria and cytokines. Emotional or psychological stress, a risk factor of periodontal disease, is transmitted by stress modulators including norepinephrine and cortisol. The aim of the present study was to investigate the effect of stress on calprotectin expression using norepinephrine and cortisol. U-937 cells, a human monocytic cell line, were incubated with norepinephrine in the presence or absence of beta- or alpha-adrenergic receptor antagonists, or with cortisol. The expression of S100A8/S100A9 mRNAs was examined by northern blotting and the amount of calprotectin was measured by enzyme-linked immunosorbent assay (ELISA). The DNA binding activity of C/EBPalpha (CCAAT enhancing binding protein), a transcription factor, was examined by electrophoretic mobility shift assay. Norepinephrine stimulated the expression of S100A8/S100A9 mRNAs via beta-adrenergic receptors in U-937 cells and significantly increased calprotectin production to about 3.6-fold that of the control. However, cortisol had no effect on calprotectin expression at the mRNA and protein levels. Norepinephrine elevated C/EBPalpha DNA binding activity, but cortisol did not increase the activity. Norepinephrine, a stress modulator, stimulated calprotectin expression in human monocytic cells. Calprotectin expression may be regulated by stress in addition to inflammatory factors.
Chie Wada -Mihara, Masatoshi Kataoka, Hiroyuki Seto, Noriko Hayashi, Jun-ichi Kido, Yasuo Shinohara and Toshihiko Nagata : High-turnover osteoporosis is induced by cyclosporin A in rats., Journal of Bone and Mineral Metabolism, Vol.24, No.3, 199-205, 2006.
(Summary)
Cyclosporin A (CsA) is used widely as an immunosuppressive agent, but it induces osteoporosis as a prominent side effect. To elucidate the mechanisms involved in CsA-induced osteoporosis, the effects of CsA on bone metabolism were investigated in a rat experimental model. Fifteen-day-old rats were fed a powdered diet containing or lacking CsA for 8-30 days. Analysis was performed by micro-computed tomography (muCT) and light microscopy to examine histomorphometric changes in rat tibiae on days 8, 16, and 30. Plasma parathyroid hormone (PTH) and osteocalcin (OCN) levels were determined by enzyme-linked immunosorbent assay (ELISA) on days 8, 16, and 30. The expression of OCN, osteopontin (OPN), and cathepsin K mRNAs in tibial bone marrow was examined by Northern blot analysis on days 8 and 16. Although no significant differences were observed in tibial length during the experimental periods, or in histomorphometric parameters on day 8, an apparent decrease in bone volume was observed in the CsA-treated group after day 16. Histologic analysis showed that the number of osteoblasts and osteoclasts on the surface of trabecular bone in the CsA-treated group had increased significantly on day 16. Plasma PTH and OCN levels in CsA-treated rats were significantly higher than those in control animals on day 8. Northern blot analysis revealed that the CsA-treated group showed an increase in the expression of OCN, OPN, and cathepsin K mRNAs on day 8 compared with the controls. These findings suggest that bone resorption in CsA-treated rats is induced by high-turnover osteoporosis and that bone remodeling activity may be activated by PTH.
Yoshitaka Kihira, Mitsuru Hashimoto, Yasuo Shinohara, Eiji Majima and Hiroshi Terada : Roles of adjoining Asp and Cys residues of first matrix-facing loop in transport activity of yeast and bovine mitochondrial ADP/ATP carriers, The Journal of Biochemistry, Vol.139, No.3, 575-582, 2006.
(Summary)
The mitochondrial ADP/ATP carrier (AAC) transports substrate by interconversion of its conformation between m- and c-states. The 1st loop facing the matrix (LM1) is extruded into the matrix in the m-state and is suggested to intrude into the mitochondrial membrane on conversion to the c-state conformation [Hashimoto, M., Majima, E., Goto, S., Shinohara, Y., and Terada, H. (1999) Biochemistry 38, 1050-1056]. To elucidate the mechanism of the translocation of LM1, we examined the effects of site-directed mutagenesis of two adjoining residues, Cys56 and Asp55 in the bovine type 1 AAC and Cys73 and Asp72 in the yeast type 2 AAC, on the substrate transport activity. We found that (i) replacement of the Cys by bulky and hydrophilic residues was unfavorable for efficient transport activity, (ii) the carboxyl groups of the Asp residues of the bovine and yeast AACs were essential and strictly position-specific, and (iii) hence, the mutation to Glu showed transport activity comparable to that of the native AACs. Based on these results, we discussed the functional role of LM1 in the transport activity of AAC.
Mari Ogino, Jun-ichi Kido, Mika Bando, Noriko Hayashi, Chie Wada -Mihara, Toshihiko Nagata, Fusanori Nishimura, Y. Soga, Shogo Takashiba, T. Kubota, M. Itagaki, Yasuko Shimada, H. Tai, Hiromasa Yoshie, Naoshi Yamazaki, Yasuo Shinohara and Masatoshi Kataoka : Alpha 2 integrin +807 polymorphism in drug-induced gingival overgrowth, Journal of Dental Research, Vol.84, No.12, 1183-1186, 2005.
(Summary)
Alpha2 integrin on fibroblasts is reported to play an important role in the induction of drug-induced gingival overgrowth, which is characterized by excessive accumulation of type I collagen in gingival connective tissue. Silent polymorphism 807 T/C within the alpha2 integrin gene is associated with high/low alpha2 integrin expression. The aim of this study was to test the hypothesis that expression of alpha2 integrin 807 T/C polymorphism correlates with drug-induced gingival overgrowth. A case-control study comparing 136 subjects taking calcium channel blockers (72 with vs. 64 without drug-induced gingival overgrowth) demonstrated that the frequency of the +807 C allele was significantly higher in the case group than in the controls (odds ratio, 3.61; 95% confidence interval, 2.14 - 6.10; P < 0.05). The present findings suggest that the alpha2 +807 C allele is one of the genetic risk factors for drug-induced gingival overgrowth.
Takenori Yamamoto, Satsuki Terauchi, Aiko Tachikawa, Kikuji Yamashita, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Two critical factors affecting the release of mitochondrial cytochrome c as revealed by studies using N, N'-dicyclohexylcarbodiimide as an atypical inducer of permeability transition, Journal of Bioenergetics and Biomembranes, Vol.37, No.5, 299-306, 2005.
(Summary)
N,N'-dicyclohexylcarbodiimide (DCCD) was earlier reported to have stimulatory effects on mitochondrial respiration and to induce mitochondrial swelling, when it was added to mitochondrial suspensions. These data seem to imply that DCCD caused the mitochondrial permeability transition (PT), but this possibility had never been investigated. In the present study, effects of DCCD on the mitochondrial structure and function were studied in detail. DCCD was found to induce mitochondrial PT in a cyclosporine A-insensitive manner. Electron microscopic analysis also supported the induction of the mitochondrial PT by DCCD. However, different from many other PT inducers, DCCD failed to cause massive release of mitochondrial cytochrome c. To understand the relationship between the induction of mitochondrial PT and the release of mitochondrial cytochrome c, we compared the actions of DCCD on mitochondrial structure and function with those of Ca2+, known as an ordinary PT inducer. As a result, two parameters considered to be critical for controlling the release of mitochondrial cytochrome c on the induction of PT were mitochondrial volume and the velocity of mitochondrial oxygen consumption.
(Keyword)
Animals / Cell Membrane / Cell Membrane Permeability / Cells, Cultured / Cytochromes c / Dicyclohexylcarbodiimide / Dose-Response Relationship, Drug / Male / Mitochondria, Liver / Rats / Rats, Wistar
Masatoshi Kataoka, Yoko Fukura, Yasuo Shinohara and Yoshinobu Baba : Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry, Electrophoresis, Vol.26, No.15, 3025-3031, 2005.
(Summary)
The mitochondrial membrane potential (DeltaPsi(m)) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of DeltaPsi(m), we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) fluorescent probe. The use of DiOC(6)(3) in the fluorometer was shown to be feasible for monitoring variations in DeltaPsi(m) in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 microM FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC(6)(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.
Satsuki Terauchi, Takenori Yamamoto, Kikuji Yamashita, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Molecular basis of morphological changes in mitochondrial membrane accompanying induction of permeability transition, as revealed by immuno-electron microscopy., Mitochondrion, Vol.5, No.4, 248-254, 2005.
(Summary)
The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), beta-subunit of F1-ATPase (F1beta), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F1beta and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.
Kazuaki Kajimoto, Hiroshi Terada, Yoshinobu Baba and Yasuo Shinohara : Essential role of citrate export from mitochondria at early differentiation stage of 3T3-L1 cells for their effective differentiation into fat cells, as revealed by studies using specific inhibitors of mitochondrial di- and tricarbodylate carriers, Molecular Genetics and Metabolism, Vol.85, No.1, 46-53, 2005.
(Summary)
1,2,3-Benzenetricarboxylate (BTA) and n-butylmalonate (BM), specific inhibitors of the mitochondrial tricarboxylate and dicarboxylate carrier, respectively, have been used to study the contribution of citrate export from mitochondria to the accumulation of fat in 3T3-L1 cells. Continuous treatment of the cells with BTA or BM for 5 days after the induction of differentiation caused a significant reduction in fat accumulation in the cells in an inhibitor concentration-dependent manner. These inhibitory effects of BTA and BM were not due to their side effects on DNA replication, since similar inhibition of fat accumulation was not observed with ordinary inhibitors of DNA replication. A similar reduction in fat accumulation was also observed when the cells were treated with BTA or BM for only 2 days just after induction of differentiation. However, interestingly, treatment of the cells with an inhibitor starting 2 days after the induction did not result in reduced fat accumulation. Furthermore, Northern analysis clearly indicated that transcript levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and adipose-type fatty acid binding protein (A-FABP) were well correlated with the levels of fat accumulation. These results clearly indicate the essential role of citrate export from the mitochondrial matrix to the cytosol at the early differentiation stage of 3T3-L1 cells for their effective differentiation into fat cells.
Yoshitaka Kihira, Eiji Majima, Yasuo Shinohara and Hiroshi Terada : Cysteine labeling studies detect conformational changes in region 106-132 of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae, Biochemistry, Vol.44, No.1, 184-192, 2005.
(Summary)
To know the structural and functional features of the cytosolic-facing first loop (LC1) including its surrounding region of the mitochondrial ADP/ATP carrier (AAC), we prepared 27 mutants, in which each amino acid residue between residues 106 and 132 of the yeast type 2 AAC (yAAC2) was replaced by a cysteine residue. For mutant preparation, we used a Cys-less AAC mutant, in which all four intrinsic cysteine residues were substituted with alanine residues, as a template [Hatanaka, T., Kihira, Y., Shinohara, Y., Majima, E., and Terada, H. (2001) Biochem. Biophys. Res. Commun. 286, 936-942]. From the labeling intensities of the membrane-impermeable SH-reagent eosin-5-maleimide (EMA), sequence Lys(108)-Phe(127) was suggested to constitute the LC1. The N-terminal half of this region (Lys(108)-Phe(115)) was suggested to change its location from the cytosol to a region close to the membrane on conversion from the c-state to the m-state in association with disruption or unwinding of its alpha-helical structure, whereas the C-terminal half region (Gly(116)-Phe(127)) was considered to extrude essentially into the cytosol, while keeping its alpha-helical structure. Hence, the conformation of m-state LC1 is greatly different from that of c-state LC1. Possibly the LC1 changes its location between the membranous region and the cytosol during ADP/ATP transport. Lys(108) in the LC1 of the yAAC2 was found to be associated with binding of the transport substrates, and its -NH(3)(+) moiety, to be of importance for the transport function. On the basis of these results, possible roles of the conformational changes of the LC1 in the transport activity are discussed.
Yoshitaka Kihira, Akihiro Iwahashi, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Twisting of the second transmembrane a-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states, Biochemistry, Vol.43, No.48, 15204-15209, 2004.
(Summary)
To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.
(Keyword)
Amino Acid Sequence / Amino Acid Substitution / Animals / Atractyloside / Binding Sites / Cattle / Cysteine / Enzyme Inhibitors / Eosine Yellowish-(YS) / Intracellular Membranes / Mitochondrial ADP, ATP Translocases / Molecular Sequence Data / Mutagenesis, Site-Directed / Protein Conformation / Protein Structure, Secondary / Protein Transport / Saccharomyces cerevisiae Proteins
Tomoyuki Yuasa, Rei Kakuhata, Kazuhiro Kishi, Toshiyuki Obata, Yasuo Shinohara, Yoshimi Bando, Keisuke Izumi, Fumiko Kajiura, Mitsuru Matsumoto and Yousuke Ebina : Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing PDGF receptor in the muscle, but does not affect blood glucose levels, Diabetes, Vol.53, No.11, 2776-2786, 2004.
(Summary)
Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.
Takenori Yamamoto, Aiko Tachikawa, Satsuki Terauchi, Kikuji Yamashita, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Multiple Effects of DiS-C3(5) on Mitochondrial Structure and Function, European Journal of Biochemistry, Vol.271, No.17, 3573-3579, 2004.
(Summary)
3,3'-Dipropyl-2,2'-thiadicarbocyanine iodide [DiS-C(3)(5)], often used as a tracer dye to assess the mitochondrial membrane potential, was investigated in detail regarding its effects on the structure and function of isolated mitochondria. As reported previously, DiS-C(3)(5) had an inhibitory effect on NADH-driven mitochondrial electron transfer. On the contrary, in the presence of inorganic phosphate, DiS-C(3)(5) showed dose-dependent biphasic effects on mitochondria energized by succinate. At higher concentrations, such as 50 micro m, DiS-C(3)(5) accelerated mitochondrial oxygen consumption. Measurements of the permeability of DiS-C(3)(5)-treated mitochondrial membranes to poly(ethylene glycol) and analysis of mitochondrial configuration by transmission electron microscopy revealed that the accelerating effect of DiS-C(3)(5) on mitochondrial oxygen consumption reflects the induction of the mitochondrial permeability transition (PT). When the mitochondrial PT was induced by DiS-C(3)(5), release of mitochondrial cytochrome c was observed, as in the case of the PT induced by Ca(2+). On the contrary, at a low concentration such as 5 micro m, DiS-C(3)(5) showed an inhibitory effect on the latent oxygen consumption by mitochondria. This effect was shown to reflect inhibition of the PT induced by a low concentration of Ca(2+). Furthermore, in the absence of inorganic phosphate, DiS-C(3)(5) caused mitochondrial swelling. Under this condition, DiS-C(3)(5) caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c.
R. Jose, asudevanpillai Biju, Yoshihisa Yamaoka, Toshimi Nagase, Yoji Makita, Yasuo Shinohara, Yoshinobu Baba and Mitsuru Ishikawa : Synthesis of CdTe Quantum Dots using a Heterogeneous Process at Low Temperature and their Optical and Structural Properties, Applied Physics. A, Materials Science & Processing, Vol.79, No.8, 1833-1838, 2004.
Masatoshi Kataoka, Sonoko Inoue, Kazuaki Kajimoto, Yasuo Shinohara and Yoshinobu Baba : Usefulness of microchip electrophoresis for reliable analyses of non-satndard DNA samples and subsequent on-chip enzymatic digestion, European Journal of Biochemistry, Vol.271, No.11, 2241-2247, 2004.
(Summary)
The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-1), error increased in a dose-dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.
(Keyword)
DNA / Deoxyribonucleases, Type II Site-Specific / Electrophoresis, Capillary / Plasmids / Reproducibility of Results
Rieko Tachibana, Naoko Ide, Yasuo Shinohara, Hideyoshi Harashima, C. Anthony Hunt and Hiroshi Kiwada : An assessment of relative transcriptional availability from nonviral vectors, International Journal of Pharmaceutics, Vol.270, No.1-2, 315-321, 2004.
(Summary)
To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our understanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanolamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression.
(Keyword)
Animals / Chloramphenicol O-Acetyltransferase / Drug Carriers / Drug Delivery Systems / Gene Expression / Gene Transfer Techniques / Genes, Reporter / Genetic Vectors / Liposomes / Mice / NIH 3T3 Cells / Plasmids / Polyethyleneimine / Time Factors / Transcription, Genetic
Akira Unami, Yasuo Shinohara, Kazuaki Kajimoto and Yoshinobu Baba : Comparison of gene expression profiles between white and brown adipose tissues of rat by microarray analysis, Biochemical Pharmacology, Vol.67, No.3, 555-564, 2004.
(Summary)
To characterize the energy metabolism in brown adipose tissue (BAT), the differences in gene expression profiles between BAT and white adipose tissue (WAT) were analyzed using a high-density cDNA microarray. RNAs isolated from two adipose tissues were hybridized to an Agilent rat cDNA Microarray that contained about 14,500 cDNA probe sets. The expression levels of 499 cDNA/ESTs were found to be at least 5-fold higher or lower in BAT than in WAT. Consistent with our previous findings, high expression levels of genes encoding uncoupling protein 1, muscle-type carnitine palmitoyltransferase and some other proteins involved in energy metabolism in BAT were found. Most of the genes encoding mitochondrial proteins, such as subunits of ATP synthase, cytochrome c oxidase, and NADH dehydrogenase, were highly expressed, reflecting possible differences in the cellular content of mitochondria between BAT and WAT. However, the expression levels of several genes encoding mitochondrial protein, such as liver mitochondrial aldehyde dehydrogenase and dicarboxylate carrier, were remarkably lower in BAT. These results may give important clues to understand the unique energy metabolism in BAT.
Kikuji Yamashita, Tomokazu Ichikawa, Takenori Yamamoto, Yoshinori Nakagawa, Hiroshi Terada and Yasuo Shinohara : Three-way effect of cyanine dye on the structure and function of mitochondria, Journal of Health Science, Vol.49, No.6, 448-453, 2003.
(Summary)
Previously, we found that the cationic cyanine dye tri-S-C_4(5) uncoupled mitochondrial oxidative phosphorylation by acting as an inducer of the mitochondrial permeability transition (PT). In the present study, the actions of cyanine dyes such as tri-S-C_4(5) and tri-S-C_7(5) on the mitochondrial structures and functions were further characterized. In the presence of inorganic phosphate (Pi), cyanine dyes were found to accelerate mitochondrial oxygen consumption that was partially sensitive to the PT inhibitor cyclosporin A (CsA). However, not only the CsA-sensitive but also CsA-insensitive acceleration of mitochondrial respiration was induced by cyanine dyes; and both of them were found to be associated with the release of mitochondrial cytochrome c. On the contrary, in the absence of Pi, moderate acceleration of respiration was induced by cyanine dyes, but this respiratory effect was not associated with the iniduction of swelling or the release of mitochondrial cytochrome c. Thus, cyanine dyes were concluded to have 3 different effects on the mitochondrial functions depending on the Pi status.
(Keyword)
mitochondria / permeability transition / cyanine dyes / release of cytochrome c / apoptosis
Kazuaki Kajimoto, Takiko Daikoku, Fumiyo Kita, Naoshi Yamazaki, Masatoshi Kataoka, Yoshinobu Baba, Hiroshi Terada and Yasuo Shinohara : PCR-select subtraction for characterization of messages differentially expressed in brown compared with white adipose tissue., Molecular Genetics and Metabolism, Vol.80, No.1-2, 255-261, 2003.
(Summary)
To understand the energy metabolism occurring in brown adipose tissue (BAT), we subtracted the messages expressed in white adipose tissue (WAT) from those in BAT. Thereby we succeeded in identifying 37 cDNA clones as being significantly expressed in BAT but not in WAT. Of these, 24 clones were found to code for mitochondrial proteins. Since BAT is well known to have a higher mitochondrial content than WAT, these results would seem to reflect simply the differences in mitochondrial content between BAT and WAT. To examine this possibility, we next measured the amount of mitochondrial DNA (mtDNA) in various rat tissues. As a result, the mtDNA copy number per cell was found to be markedly different among the tissues analyzed, and the highest value of about 5.3x10(4) copies per cell was observed with the rat brain. BAT showed a value similar to that of brain, but this value was only about 3.5-fold higher than that for WAT. Since observed differences in mitochondrial content between BAT and WAT was smaller than those observed with transcript levels of proteins, we conclude that the observed differences in the transcript levels of certain proteins between BAT and WAT reflect the functional differences between BAT and WAT, and do not reflect the differences in mitochondrial content between BAT and WAT.
Radwan Mohamad Almofti, Tomokazu Ichikawa, Kikuji Yamashita, Hiroshi Terada and Yasuo Shinohara : Silver ion induces a cyclosporine a-insensitive permeability transition in rat liver mitochondria and release of apoptogenic cytochrome C, The Journal of Biochemistry, Vol.134, No.1, 43-49, 2003.
(Summary)
Various reagents are known to open the mitochondrial permeability pore (PTP) and induce a permeability transition (PT), releasing apoptogenic proteins from the intermembrane space and triggering apoptosis. In this study, we examined the effect of Ag(+), a known cytotoxic sulfhydryl-reactive heavy metal, on isolated rat liver mitochondria. The following results were obtained: (1) Upon addition, Ag(+) instantly induced mitochondrial swelling and acceleration of respiration. (2) Cyclosporine A, a specific inhibitor of classical PT, was ineffective against the effect of Ag(+), indicating that silver ions induced non-classic PT. (3) Sulfhydryl reagents such as reduced glutathione completely inhibited the effects of Ag(+) on the mitochondria. (4) Experimental results using polyethylene glycol indicated that Ag(+) induced opening of a pore in the inner mitochondrial membrane, which could be PTP of another open state or a distinct pore. (5) Electron microscopic analysis of mitochondria treated with Ag(+) showed a novel mitochondrial configuration that was apparently different from that of normal mitochondria or Ca(2+)-treated mitochondria. (6) Ag(+) also induced the release of apoptogenic cytochrome c in a CsA-insensitive but GSH-sensitive manner. These results suggest that Ag(+) promotes a nonclassical permeability increase in the mitochondrial inner membrane that is clearly distinguishable from the classical PT and releases apoptogenic cytochrome c in a classical PT-independent manner.
Naoshi Yamazaki, Yasuhisa Yamanaka, Yoshiko Hashimoto, Takayuki Hiramatsu, Yasuo Shinohara and Hiroshi Terada : The gene encoding muscle-type carnitine palmitoyltransferase I: comparison of the 5'-upstream region of human and rodent genes, The Journal of Biochemistry, Vol.133, No.4, 523-532, 2003.
(Summary)
Muscle-type carnitine palmitoyltransferase I (M-CPTI) is the key enzyme for fatty acid beta-oxidation in heart and skeletal muscles and in adipose tissue. So far, M-CPTI mRNA has been detected in white adipocytes from epididymal fat pads of rats and humans, but not in mouse adipocytes. To characterize the gene expression of M-CPTI in mice, we isolated the genomic DNA encoding mouse M-CPTI and determined its transcription initiation site. As a result, the mouse M-CPTI gene seemed to have multiple initiation sites, as in the case of the rat and human genes. Furthermore, the conserved nucleotide sequence of the response element for peroxisome proliferators was shown to exist in the upstream of the mouse gene as in that of the rat and human genes. From these observations, we suggest that the anomalous expression of M-CPTI in mouse adipocytes reported previously may be regulated by factors other than peroxisome proliferators. Previously, we reported that there were transcripts containing regions of both CK/EK-beta and M-CPTI genes in humans. In this study, we found that such transcripts also exist in rodents and that the amounts of the transcripts containing regions of both of these genes did not depend on the expression level of CK/EK-beta.
Kazuaki Kajimoto, Takiko Daikoku, Naoshi Yamazaki, Hiroshi Terada and Yasuo Shinohara : Expression profiles of three isoforms of inositol 1,4,5-trisphosphate receptor in brown adipose tissue of the rat., Biochemical Pharmacology, Vol.65, No.6, 995-998, 2003.
(Summary)
The thermogenic function of brown adipose tissue (BAT) is known to be mainly regulated by a signal transduction cascade via beta-adrenoceptor. However, recent studies indicated that the alpha-adrenoceptor and its downstream signal transduction cascade, causing elevation of the cellular Ca(2+) level, are also important for the regulation of this function of BAT. In the present study, expression profiles of 3 isoforms of the inositol 1,4,5-trisphosphate (IP(3)) receptor, known as one of the major components of the machinery regulating the intracellular Ca(2+) concentration in the BAT of rats, were investigated by Northern analysis. Of these three IP(3) receptor isoforms, the type 2 one was found to be the most abundant of the three in BAT. Furthermore, when rats were exposed to the cold, under which condition the thermogenic function of BAT is known to be stimulated, the expression levels of types 1 and 2 isoforms of IP(3) receptor were remarkably elevated. The results of Western analysis supported the predominant expression of the type 2 isoform in BAT. However, different from the results of Northern analysis, the expression levels of types 1 and 2 isoforms of IP(3) receptor protein in BAT were not influenced by exposure of the animals to the cold.
(Keyword)
Adipose Tissue, Brown / Animals / Blotting, Northern / Calcium Channels / Energy Metabolism / Gene Expression Profiling / Inositol 1,4,5-Trisphosphate Receptors / Male / Protein Isoforms / Rats / Rats, Wistar / Receptors, Cytoplasmic and Nuclear
Mohamad Radwan Almofti, Hideyoshi Harashima, Yasuo Shinohara, Almofti Ammar, Yoshinobu Baba and Hiroshi Kiwada : Cationic liposome-mediated gene delivery:Biophysical study and mechanism of internalization Arch, Archives of Biochemistry and Biophysics, Vol.410, No.2, 246-253, 2003.
(Summary)
To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.
Mohamad Radwan Almofti, Hideyoshi Harashima, Yasuo Shinohara, Ammar Almofti, Wenhao Li and Hiroshi Kiwada : Lipoplex size determines lipofection efficiency with or without serum., Molecular Membrane Biology, Vol.20, No.1, 35-43, 2003.
(Summary)
In order to identify factors affecting cationic liposome-mediated gene transfer, the relationships were examined among cationic liposome/DNA complex (lipoplex)-cell interactions, lipoplex size and lipoplex-mediated transfection (lipofection) efficiency. It was found that lipofection efficiency was determined mainly by lipoplex size, but not by the extent of lipoplex-cell interactions including binding, uptake or fusion. In addition, it was found that serum affected mainly lipoplex size, but not lipoplex-cell interactions, which effect was the major reason behind the inhibitory effect of serum on lipofection efficiency. It was concluded that, in the presence or absence of serum, lipoplex size is a major factor determining lipofection efficiency. Moreover, in the presence or absence of serum, lipoplex size was found to affect lipofection efficiency by controlling the size of the intracellular vesicles containing lipoplexes after internalization, but not by affecting lipoplex-cell interactions. In addition, large lipoplex particles showed, in general, higher lipofection efficiency than small particles. These results imply that, by controlling lipoplex size, an efficient lipid delivery system may be achieved for in vitro and in vivo gene therapy.
(Keyword)
Cationic Liposomes / Dna / Lipofection / Fusion / Uptake
Yasuo Shinohara, Radwan Mohamad Almofti, Takenori Yamamoto, Taro Ishida, Fumiyo Kita, Hideki Kanzaki, Masakatsu Ohnishi, Kikuji Yamashita, Shigeomi Shimizu and Hiroshi Terada : Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin, European Journal of Biochemistry, Vol.269, No.21, 5224-5230, 2002.
(Summary)
To examine whether valinomycin induces a mitochondrial permeability transition (PT), we investigated its effects on mitochondrial functions under various conditions. The acceleration of mitochondrial respiration and swelling, induced by valinomycin, were found to be insensitive to inhibitors of the ordinary PT, indicating that valinomycin does not induce the ordinary PT. Results of experiments using mitochondria isolated from transgenic mice expressing human bcl-2 also supported this conclusion. Furthermore, evidence for induction of PT pores by valinomycin was not obtained by either electron microscopic analysis of mitochondrial configurations or by measurement of the permeability of the inner mitochondrial membrane by use of polyethylene glycol. However, valinomycin did induce a significant release of cytochrome c, and thus it may be a nice tool to study the processes of mitochondrial cytochrome c release.
Mari Tabuchi, Mami Hino, Yasuo Shinohara and Yoshinobu Baba : Cell-free protein synthesis on a microchip, Proteomics, Vol.2, No.4, 430-435, 2002.
(Summary)
We evaluated the expression of various proteins by using a cell-free protein synthesis system with a rapid translation system, a microtube, and a microchip technique. Protein expression was successfully achieved with a microfabricated reaction chamber on a plastic chip. Proteins were expressed effectively by use of expression vectors for T7 RNA polymerase (pET) instead of a plasmid for in vitro expression vector, which is recommended by the manufacture of the rapid translation system. Expression of the proteins depended on the type of proteins chosen. Two mammalian proteins were synthesized simultaneously with two expression vectors of pET species. Effective application of the cell-free protein synthesis system will enable miniaturization of protein synthesis and mediation between the transcriptome and proteome.
(Keyword)
Animals / Cell-Free System / DNA-Directed RNA Polymerases / Humans / Miniaturization / Protein Array Analysis / Protein Biosynthesis / Proteins / Proteome / Viral Proteins
Rieko Tachibana, Hideyoshi Harashima, Naoko Ide, Sachiko Ukitsu, Yasuko Ohta, Norio Suzuki, Hiroshi Kikuchi, Yasuo Shinohara and Hiroshi Kiwada : Quantitative Analysis of Correlation between Nunber of Nuclear Plasmids and Gene Expression Activity after Transfection with Cationic Liposomes, Pharmaceutical Research, Vol.19, No.4, 377-381, 2002.
(Summary)
A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed. AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification. Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose. These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.
(Keyword)
Animals / Cations / Cell Nucleus / Dose-Response Relationship, Drug / Drug Delivery Systems / Drug Evaluation, Preclinical / Gene Expression Regulation / Liposomes / Plasmids / Polymerase Chain Reaction / Rats / Transfection / Tumor Cells, Cultured
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12033367
Rieko Tachibana, Hideyoshi Harashima, Tatsuhiro Ishida, Yasuo Shinohara, Mari Hino, Hiroshi Terada, Yoshinobu Baba and Hiroshi Kiwada : Effect of Cationic Liposomes in an in Vitro Transcription and Translation System, Biological & Pharmaceutical Bulletin, Vol.25, No.4, 529-531, 2002.
(Summary)
The effects of cationic liposomes complexed with plasmid DNA on the process of transcription was examined using a recently developed rapid cell free translation system. The findings indicate that the liposome itself inhibited the process when the ratio of DNA/liposome typically used in transfection studies was used.
Mami Hino, Yasuo Shinohara, Kazuaki Kajimoto, Hiroshi Terada and Yoshinobu Baba : Requirement of continuous transcription for the synthesis of sufficient amounts of protein by a cell-free rapid translation system, Protein Expression and Purification, Vol.24, No.2, 255-259, 2002.
(Summary)
To understand the key processes of cell-free protein synthesis, the synthesis of adipose-type fatty acid binding protein (A-FABP) by a rapid translation system was examined under various conditions. The synthesis of A-FABP was achieved by using an expression vector of A-FABP containing a T7 promoter. However, synthesis of A-FABP was not observed when an RNA fragment corresponding to the open reading frame of A-FABP was used in the reaction instead of the expression vector. Northern analysis revealed that the RNA that was added to the reaction mixture promptly underwent degradation. On the contrary, when the expression vector of A-FABP was employed, a strong RNA signal was observed over the entire incubation period. Thus, a continuous supply of RNA is needed in order to account for its loss via degradation to achieve the synthesis of reasonable amounts of A-FABP. Furthermore, the effect of continuous exchange of reaction mixture was also evaluated by measurement of the amount of synthesized A-FABP.
Eiji Majima, Masashi Takeda, Satomi Miki, Yasuo Shinohara and Hiroshi Terada : Close location of the first loop to the third loop of the mitochondrial ADP/ATP carrier deduced from cross-linking catalyzed by copper-o-phenanthroline of the solubilized carrier with Triton X-100, The Journal of Biochemistry, Vol.131, No.3, 461-468, 2002.
Naoshi Yamazaki, Yasuo Shinohara, Kayo Tanida and Hiroshi Terada : Structural properties of mammalian mitochondrial ADP/ATP carriers: identification of possible amino acids that determine functional differences in its isoforms., Mitochondrion, Vol.1, No.4, 371-379, 2002.
(Summary)
The structural properties of isoforms of the mitochondrial ADP/ATP carrier expressed in mammals were characterized in order to understand their possible functional differences. To accomplish this, the cDNA clone of the bovine type 2 isoform was isolated and characterized. We also extensively explored the rat type 3 isoform, but it was not detected. We next compared the amino acid sequences of the ten ADP/ATP carriers, which are expressed in mammals. As a result, amino acids at positions 45, 147 and 164 were found to show strict isoform dependency regardless of species differences. Thus, they are expected to determine functional differences in the isoforms of the ADP/ATP carrier.
Takashi Hatanaka, Yoshitaka Kihira, Yasuo Shinohara, Eiji Majima and Hiroshi Terada : Characterization of loops of the yeast mitochondrial ADP/ATP carrier facing the cytosol by site-directed mutagenesis, Biochemical and Biophysical Research Communications, Vol.286, No.5, 936, 2001.
(Summary)
To characterize structural features of the regions of the yeast type 2 ADP/ATP carrier (yAAC2) facing the cytosol, we prepared its Cys-less mutant, in which all four cysteine residues were replaced by alanine residues. The Cys-less mutant functioned like native yAAC2, showing that the cysteine residues are not essential. We then prepared cysteine mutants by substituting Ser(21) in the putative N-terminal region, Ala(124) and Ser(222) in the first and second loops facing cytosol, respectively, and Leu(312) in the C-terminal region of the Cys-less mutant for cysteine and examined the labeling of the substituted cysteine residues of the mutants with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) from the cytosol. EMA labeled all the mutants, showing that all regions containing mutated residues faced the cytosolic side. The effects of transport inhibitors on EMA labeling were also examined. From the results, the location and conformation of the region around mutated residues were discussed.
(Keyword)
Adenosine Diphosphate / Alanine / Anti-Bacterial Agents / Blotting, Western / Bongkrekic Acid / cell division / Cysteine / Cytosol / DNA / DNA Primers / Electrophoresis, Polyacrylamide Gel / Eosine Yellowish-(YS) / Fluorescent Dyes / mitochondria / Mitochondrial ADP, ATP Translocases / Models, Biological / Mutagenesis, Site-Directed / Mutation / Protein Structure, Tertiary / Protein Transport / Saccharomyces cerevisiae / Saccharomyces cerevisiae Proteins / Serine
Takashi Hatanaka, Mitsuru Hashimoto, Eiji Majima, Yasuo Shinohara and Hiroshi Terada : Significant effect of the N-terminal region of the mitochondrial ADP/ATP carrier on its efficient expression in yeast mitochondria, The Journal of Biological Chemistry, Vol.276, No.31, 28881-28888, 2001.
(Summary)
The low-level expression of the bovine heart mitochondrial ADP/ATP carrier (bovine type 1 ADP/ATP carrier (bAAC1)) in the yeast mitochondrial membrane is significantly improved by replacement of its N-terminal region with corresponding regions of the yeast type 1 and 2 carriers (yAAC1 and yAAC2) (Hashimoto, M., Shinohara, Y., Majima, E., Hatanaka, T., Yamazaki, N., and Terada, H. (1999) Biochim. Biophys. Acta 1409, 113--124). To understand why the bAAC1 chimeras were highly expressed in yeast mitochondria, we examined the effects of the length and sequence of the N-terminal region extending into the cytosol on the expression of bAAC1 and yAAC2 derivatives in yeast mitochondria. For this, their N-terminal regions were replaced with peptide fragments of various lengths and sequences derived from those of bAAC1, yAAC1, and yAAC2. We found that a specific amino acid sequence and a definite length of the N-terminal region of yAAC2 were required for high expression of bAAC1 and yAAC2 in yeast mitochondria. We also examined the steady-state transcript levels and expression of these derivatives in whole yeast cells. Based on our results, we discuss the role of the N-terminal region in efficient expression of bAAC1 and yAAC2 in yeast mitochondria.
Eiji Majima, Mayumi Ishida, Satomi Miki, Yasuo Shinohara and Hiroshi Terada : Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate, --- Roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport ---, The Journal of Biological Chemistry, Vol.276, No.13, 9792-9799, 2001.
(Summary)
The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.
(Keyword)
Adenosine Diphosphate / Adenosine Triphosphate / Amino Acid Sequence / Animals / Binding Sites / Cattle / Cell Membrane / Chromatography, High Pressure Liquid / Cytosol / DNA / Dose-Response Relationship, Drug / Fluorescein-5-isothiocyanate / Fluorescent Dyes / Hydrogen-Ion Concentration / Lysine / Mitochondria / Models, Chemical / Molecular Sequence Data / Phosphates / Protein Binding / Protein Conformation / Sequence Analysis, Protein / Temperature / Time Factors
Yasuo Shinohara, Takiko Daikoku, Kazuaki Kajimoto, Atsushi Shima, Naoshi Yamazaki and Hiroshi Terada : Expression of NAD(+)-dependent isocitrate dehydrogenase in brown adipose tissue., Biochemical and Biophysical Research Communications, Vol.281, No.3, 634-638, 2001.
(Summary)
cDNA clones significantly expressed in brown adipose tissue (BAT) but not in white adipose tissue (WAT) of rats were isolated by use of a PCR-select cDNA subtraction kit. Of the isolated clones, structural features of two of them, 2-58 and 2-67, were studied in detail. The results indicated that these clones were cDNAs encoding alpha- and beta-subunits of rat NAD(+)-dependent isocitrate dehydrogenase (NAD(+)-ICDH). Previous biochemical study suggested the importance of NAD(+)-ICDH in metabolism in BAT; however, transcript levels of individual subunits of this enzyme in BAT had never been analyzed. In the present study, using these newly isolated cDNAs, we clearly demonstrate that the expression of three subunits of NAD(+)-ICDH was the most remarkable in BAT among the various tissues analyzed.
(Keyword)
Adipose Tissue, Brown / Animals / Base Sequence / DNA, Complementary / Isocitrate Dehydrogenase / Molecular Sequence Data / NAD / Polymerase Chain Reaction / Rats / Sequence Homology, Amino Acid / Sequence Homology, Nucleic Acid
Shigeomi Shimizu, Yosuke Matsuoka, Yasuo Shinohara, Yoshihiro Yoneda and Yoshihide Tsujimoto : Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells, The Journal of Cell Biology, Vol.152, No.2, 237-250, 2001.
(Summary)
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.
(Keyword)
Amino Acid Sequence / Animals / Antibodies / Antibodies, Monoclonal / apoptosis / Apoptosis Regulatory Proteins / BH3 Interacting Domain Death Agonist Protein / Carrier Proteins / Cytochrome c Group / Erythrocytes / Etoposide / HeLa Cells / Humans / Ion Channels / Mammals / Membrane Proteins / mitochondria / Mitochondria, Liver / Molecular Sequence Data / Paclitaxel / Porins / Protein Conformation / Proteins / Proto-Oncogene Proteins / Proto-Oncogene Proteins c-bcl-2 / Rats / Rats, Inbred Strains / Recombinant Proteins / Staurosporine / Transfection / Voltage-Dependent Anion Channels / bcl-2 Homologous Antagonist-Killer Protein / bcl-2-Associated X Protein
Yasuo Shinohara, Mami Hino, Taro Ishida, Yasuhisa Yamanaka and Hiroshi Terada : Growth condition-dependent synchronized changes in transcript levels of type II hexokinase and type 1 glucose transporter in tumor cells, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol.1499, No.3, 242-248, 2001.
(Summary)
Transcript levels of hexokinase (HK) isozymes and glucose transporter (GLUT) isoforms in RNA samples of AH130 cells obtained from dish cultures and ascites were evaluated in a quantitative manner. In AH130 cells cultured in dishes, HKI and HKII were expressed at a similar level, but HKIII and HKIV were not. GLUT1 and GLUT3 were also expressed, and messages of these two isoforms represented 27% and 71%, respectively, of the total GLUT messages. A faint signal of GLUT2 was also observed. On the contrary, in cells grown as ascites, the transcript of HKII was dominant, and its level was about 15-fold over that of dish-cultured AH130 cells. Transcript levels of GLUT1 and GLUT3 were 4.5- and 2-fold, respectively, higher than those in dish-cultured cells. Thus, GLUT1 was more susceptible to changes in culture conditions than GLUT3. Based on these results, we concluded that the change in growth conditions caused synchronized changes in the transcript levels of HKII and GLUT1 in AH130 cells. However, such marked changes in the transcript levels of HKII and GLUT1 were not observed when AH130 cells were cultured in dishes under a hypoxic condition, indicating that the observed changes were not solely attributable to the difference in oxygen concentration between the ascites and cell culture conditions. Accordingly, other factors such as growth factors may be responsible for this difference in levels of HKII and GLUT1 between the two growth conditions.
(Keyword)
Animals / Base Sequence / Cell Division / Cell Hypoxia / DNA Primers / Glucose Transporter Type 1 / Hexokinase / Isoenzymes / Liver Neoplasms, Experimental / Male / Monosaccharide Transport Proteins / Protein Isoforms / RNA, Messenger / RNA, Neoplasm / Rats / Tumor Cells, Cultured
Naoshi Yamazaki, Yasuo Shinohara, Kazuaki Kajimoto, Masayuki Shindou and Hiroshi Terada : Novel expression of equivocal messages containing both regions of choline/ethanolamine kinase and muscle type carnitine palmitoyltransferase I., The Journal of Biological Chemistry, Vol.275, No.41, 31739-31746, 2000.
(Summary)
For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.
Yasuo Shinohara, Taro Ishida, Mami Hino, Naoshi Yamazaki, Yoshinobu Baba and Hiroshi Terada : Characterization of porin isoforms expressed in tumor cells., European Journal of Biochemistry, Vol.267, No.19, 6067-6073, 2000.
(Summary)
Mitochondria from malignant tumor cell lines show a higher capability for hexokinase binding than those from normal liver. To explore possible differences in hexokinase binding sites of mitochondria between tumor cells and normal liver, we characterized porin isoforms expressed in tumor cells. Cloning experiments on the three porin isoforms, VDAC1, VDAC2 and VDAC3 from malignant tumor cell line AH130 clearly showed that their primary structures were completely identical to those of the corresponding VDACs of normal liver cells. Possible expression of the fourth porin isoform in AH130 cells was excluded by degenerate primer-based RT-PCR. However, the transcript levels of the three VDAC isoforms in AH130 cells were significantly higher than those in normal liver. These results suggest that the high hexokinase-binding capability of malignant tumor cell mitochondria was not due to any structural difference, but due to a quantitative difference in binding sites.
Shigeomi Shimizu, Yasuo Shinohara and Yoshihide Tsujimoto : Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator, Oncogene, Vol.19, No.38, 4309-4318, 2000.
(Summary)
Mitochondria play an essential role in apoptosis by releasing apoptogenic molecules such as cytochrome c and AIF, and some caspases, which are all regulated by Bcl-2 family proteins. Pro-apoptotic Bax and Bak have been shown to induce cytochrome c release and loss of membrane potential (Deltapsi) leading to AIF release in the isolated mitochondria. We have previously shown that Bax and Bak open the voltage-dependent anion channel (VDAC) allowing cytochrome c to pass through the channel, and Bcl-xL closes the channel. However, it has been reported that it is adenine nucleotide translocator (ANT) with which Bax/Bcl-xL interacts that modulate the channel activity. Here, we investigated the role of ANT and VDAC in the changes of isolated mitochondria triggered by Bax and by chemicals that induce permeability transition (PT). In rat and yeast mitochondria, Bax did not affect the ADP/ATP exchange activity of ANT. VDAC-deficient but not ANT-deficient yeast mitochondria showed resistance to cytochrome c release, Deltapsi loss, and swelling caused by Bax and PT inducers. Bcl-xL showed similar inhibition of all these changes in ANT-deficient and wild type yeast mitochondria. Furthermore, Bax induces cytochrome c release in wild type yeast cells but not VDAC1-deficient yeast cells. These data indicate that VDAC, but not ANT, is essential for apoptotic mitochondrial changes. The data also indicate that Bcl-xL and Bax possess an ability to regulate mitochondrial membrane permeability independently of other Bcl-2 family members.
(Keyword)
Adenosine Triphosphate / Animals / Apoptosis / Cytochrome c Group / Ethanol / Male / Membrane Potentials / Mitochondria / Mitochondrial ADP, ATP Translocases / Porins / Proto-Oncogene Proteins / Proto-Oncogene Proteins c-bcl-2 / Rats / Voltage-Dependent Anion Channel 1 / Voltage-Dependent Anion Channels / Yeasts / bcl-2-Associated X Protein / bcl-X Protein
Takiko Daikoku, Yasuo Shinohara, Atsushi Shima, Naoshi Yamazaki and Hiroshi Terada : Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure., Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1457, No.3, 263-272, 2000.
(Summary)
To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of beta-adrenergic receptor (beta-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of beta(1)-AR and C/EBPbeta in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.
Mitsuru Hashimoto, Eiji Majima, Takashi Hatanaka, Yasuo Shinohara, Masakatsu Onishi, Satoru GOTO and Hiroshi Terada : Irreversible extrusion of the first loop facing the matrix of he bovine mitochondrial ADP/ATP carrier by labeling the Cys56 residue with the SH-reagent methyl methanethiosulfonate, The Journal of Biochemistry, Vol.127, No.3, 443-449, 2000.
122.
Mitsuru Hashimoto, Eiji Majima, Satoru GOTO, Yasuo Shinohara and Hiroshi Terada : Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deducing from intermolecular cross-linking of Cys56 residues by bi-functional dimaleimides, Biochemistry, Vol.38, No.3, 1050-1056, 1999.
123.
Mitsuru Hashimoto, Yasuo Shinohara, Eiji Majima, Takashi Hatanaka, Naoshi Yamazaki and Hiroshi Terada : Expression of the bovine heart mitochondrial ADP/ATP carrier in yeast mitochondria: significantly enhanced expression by replacement of the N-terminal region of the bovine carrier by the corresponding regions of the yeast carriers., Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1409, No.3, 113-124, 1999.
(Summary)
To characterize the transport mechanism mediated by the mammalian mitochondrial ADP/ATP carrier (AAC), we tried to express bovine heart mitochondrial AAC (bhAAC) in Saccharomyces cerevisiae. The open reading frame of the bhAAC was introduced into the haploid strain WB-12, in which intrinsic AAC genes were disrupted. Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane. For improvement of bhAAC expression in WB-12, we introduced DNA fragments encoding chimeric bhAACs, in which the N-terminal region of the bhAAC extending into the cytosol was replaced by the corresponding regions of the type 1 and type 2 yeast AAC isoforms (yAAC1 and yAAC2). These transformants grew well, and the amounts of the chimeric bhAACs in their mitochondria were as high as that of yAAC2. The carriers expressed showed essentially the same ADP transport activities as that of AAC in bovine heart mitochondria.
Yasuo Shinohara, Shizuko Bandou, Shinichi Kora, Seiichiro Kitamura, Shuuji Inazumi and Hiroshi Terada : Cationic uncouplers of oxidative phosphorylation are inducers of mitochondrial permeability transition, FEBS Letters, Vol.428, No.1-2, 89-92, 1998.
125.
Yasuo Shinohara, Kenji Yamamoto, Katsuyuki Inoo, Naoshi Yamazaki and Hiroshi Terada : Quantitative determinations of the steady state transcript levels of hexokinase isozymes and glucose transporter isoforms in normal rat tissues and the malignant tumor cell line AH130., Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1368, No.1, 129-136, 1998.
(Summary)
The steady state transcript levels of the four hexokinase (HK) isozymes and four glucose transporter (GLUT) isoforms were determined quantitatively by Northern analysis of RNA samples from rat tissues using synthetic fragments of the RNAs encoding the HK isozymes and GLUT isoforms. Results showed that the levels of HK isozyme transcripts were low in rat tissues, the level of that most highly expressed, the type I isozyme (HKI), in the brain being 0.025% of the total poly(A)+ RNA. A good correlation was found between the reported HK activities and the total amounts of transcripts encoding all HK isozymes in various tissues, showing that the HK activities in tissues can be estimated from the total amount of transcripts encoding HK isozymes. The proposed associated expressions of HK isozymes and GLUT isoforms in particular tissues were confirmed at their transcript levels. The steady state transcript levels of type II HK and the type 1 GLUT isoform in the malignant tumor cell line AH130 were also determined quantitatively.
(Keyword)
Animals / Electrophoresis, Agar Gel / Hexokinase / Isoenzymes / Monosaccharide Transport Proteins / RNA, Messenger / Rats / Tumor Cells, Cultured
Takiko Daikoku, Yasuo Shinohara, Atsushi Shima, Naoshi Yamazaki and Hiroshi Terada : Dramatic enhancement of the specific expression of the heart-type fatty acid binding protein in rat brown adipose tissue by cold exposure., FEBS Letters, Vol.410, No.2-3, 383-386, 1997.
127.
Naoshi Yamazaki, Yasuhisa Yamanaka, Yoshiko Hashimoto, Yasuo Shinohara, Atsushi Shima and Hiroshi Terada : Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I., FEBS Letters, Vol.409, No.3, 401-406, 1997.
128.
Yasuo Shinohara, Ikuko Sagawa, Junji Ichihara, Kenji Yamamoto, Kiyoshi Terao and Hiroshi Terada : Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1319, No.2-3, 319-330, 1997.
(Summary)
We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.
Kozo Shishido, Shigenori Haruna, Chisato Yamamura, Hiromi Iitsuka, Hisao Nemoto, Yasuo Shinohara and Masayuki Shibuya : Synthesis and evaluation of the hybrid molecules possessing DNA-cleaving activity, Bioorganic & Medicinal Chemistry Letters, Vol.7, No.20, 2617-2622, 1997.
130.
Naoshi Yamazaki, Yasuo Shinohara, Atsushi Shima, Yasuhisa Yamanaka and Hiroshi Terada : Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I., Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Vol.1307, No.2, 157-161, 1996.
(Summary)
With a cDNA probe encoding rat muscle type carnitine palmitoyltransferase I (CPTI), we isolated cDNA and genomic clones encoding the human homologue and deduced the primary structure of human muscle type CPTI. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.
Yasuo Shinohara, Akira Unami, Midori Teshima, Hitomi Nishida and Karel Dam van : Inhibitory effect of Mg2+ on the protonophoric activity of palmitic acid, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1228, No.2-3, 229-234, 1995.
(Summary)
To discriminate whether fatty acids are uncouplers that cause acceleration of State-4 respiration, associated with a decrease in the protonmotive force, or decouplers that increase respiration without associated decrease in the protonmotive force, we examined the effect of palmitate on functions of rat-liver mitochondria under various conditions. We found that palmitate itself increases State-4 respiration, releases oligomycin-inhibited State-3 respiration, inhibits ATP synthesis and ATP<->Pi exchange reaction, and increases H+ permeability in mitochondrial and model bilayer phospholipid membranes. Thus, palmitate is a classical uncoupler of oxidative phosphorylation. However, these effects were inhibited by Mg2+, due to rapid formation of a stable complex between palmitate and Mg2+.
Eiji Majima, Satoru GOTO, Hitoshi Hori, Yasuo Shinohara, Yeong-Man Hong and Hiroshi Terada : Stablilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1243, No.3, 336-342, 1995.
(Summary)
The stabilities of the SH-reagent eosin-5-maleimide (EMA) and its adducts with the SH-compounds L-cysteine, N-acetyl-L-cysteine and glutathione (reduced form) were studied under various conditions in comparison with those of the adducts of N-ethylmaleimide (NEM). Studies by reversed-phase high performance liquid chromatography and mass spectrometry showed that EMA was less stable than NEM at neutral and moderately alkaline pH values. EMA formed a succinimide-type adduct with SH-compounds, and then underwent further modification by nucleophilic attack of OH- or an amino group. The succinimide-type adducts with acetylcysteine and glutathione were converted to open-type adducts, in which the succinimide ring was cleaved, whereas the adduct with cysteine was modified to a thiazine-type adduct. Kinetic analyses showed that these open-type and thiazine-type adducts were readily formed and were stable at moderately alkaline pH values such as pH 8.0 or 9.0.
(Keyword)
Acetylcysteine / Chromatography, High Pressure Liquid / Cysteine / Drug Stability / Eosine Yellowish-(YS) / Ethylmaleimide / Glutathione / Hydrogen-Ion Concentration / Kinetics / mass spectrometry / Spectrometry, Fluorescence / Spectrometry, Mass, Fast Atom Bombardment / Succinimides / Sulfhydryl Compounds
Naoshi Yamazaki, Yasuo Shinohara, Atsushi Shima and Hiroshi Terada : High expression of a novel carnitine palmitoyltransferase I like protein in rat brown adipose tissue and heart: isolation and characterization of its cDNA clone., FEBS Letters, Vol.363, No.1-2, 41-45, 1995.
134.
Junji Ichihara, Yasuo Shinohara, Kentaro Kogure and Hiroshi Terada : Nucleotide sequence of the 5'-flanking region of the rat type II hexokinase gene, Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Vol.1260, No.3, 365-368, 1995.
(Summary)
In a previous study, we found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat hepatoma AH130 cells. To determine the molecular mechanism of transcriptional control of the type II hexokinase gene, we examined the nucleotide sequence of its 5'-flanking region and analyzed putative transcription factor binding sites. We also examined the type II hexokinase promoter activity by the chloramphenicol acetyltransferase (CAT) assay.
Eiji Majima, Yasuo Shinohara, Nana Yamaguchi, Yeong-Man Hong and Hiroshi Terada : Importance of loops of mitochondrial ADP/ATP carrier for its transport activity deduced from reactivities of its cysteine residues with the sulfhydryl reagent eosin-5-maleimide, Biochemistry, Vol.33, No.32, 9530-9536, 1994.
136.
Atsushi Shima, Yasuo Shinohara, Kyoko Doi and Hiroshi Terada : Normal differentiation of rat brown adipocytes in primary cultute judged by their expressions of uncoupling protein and the physiological isoform of glucose transporters, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol.1223, No.1, 1-8, 1994.
(Summary)
We examined the effects of dexamethasone (DEX) on the expressions of key proteins concerned with energy metabolism in brown adipocytes during their differentiation in primary culture. Transcripts of the uncoupling protein (UCP), lipoprotein lipase (LPL) and CCAAT enhancer binding protein alpha (C/EBP alpha) genes were observed in brown adipocytes cultured in the presence of insulin and thyroid hormones but in the absence of DEX. However, the mRNA level of UCP decreased with the culture period after confluence, and significant mRNA encoding type-1 glucose transporter (GLUT1) was detected in brown adipocytes cultured without DEX, whereas type-4 glucose transporter (GLUT4) was predominantly expressed in mature brown adipocytes in vivo. In contrast, DEX added after confluence consistently elevated the mRNA levels of UCP, LPL and C/EBP alpha, and repressed the level of GLUT1 in a manner synchronized with increase in the level of GLUT4. Therefore, it is concluded that DEX as well as insulin and thyroid hormones is essential for differentiation of brown adipose precursor cells into mature cells that are similar to brown adipocytes in vivo.
(Keyword)
Adipocytes / Adipose Tissue, Brown / Animals / Carrier Proteins / Cell Differentiation / Cells, Cultured / Dexamethasone / Dose-Response Relationship, Drug / Female / Ion Channels / Lipoprotein Lipase / Male / Membrane Proteins / Mitochondrial Proteins / Monosaccharide Transport Proteins / Rats / Rats, Wistar / Uncoupling Agents
Yasuo Shinohara, Kenji Yamamoto, Kentaro Kogure, Junji Ichihara and Hiroshi Terada : Steady state transcript levels of the type II hexokinase and type 1 glucose transporter in human tumor cell line, Cancer Letters, Vol.82, No.1, 27-32, 1994.
138.
Eiji Majima, Haruhiko Koike, Yeong-Man Hong, Yasuo Shinohara and Hiroshi Terada : Characterization of cysteine residues of mitochondrial ADP/ATP carrier with the SH-reagents eosin 5-maleimide, The Journal of Biological Chemistry, Vol.268, No.29, 22181-22187, 1993.
139.
Yasuo Shinohara, Makio Kamida, Naoshi Yamazaki and Hiroshi Terada : Isolation and characterization of cDNA clones and a genomic clone encoding rat mitochondrial adenine nucleotide translocator., Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1152, No.1, 192-196, 1993.
(Summary)
Two cDNA clones encoding rat mitochondrial adenine nucleotide translocator were isolated from libraries constructed from mRNAs of heart and liver. These two clones corresponded to the heart-skeletal muscle type (ANT1) and fibroblast type (ANT2), respectively. A genomic clone encoding rat ANT1 was also isolated and characterized.
Kentaro Kogure, Yasuo Shinohara and Hiroshi Terada : Evolution of the type II hexokinase gene by duplication and fusion of the glucokinase gene with conservation of its organization, The Journal of Biological Chemistry, Vol.268, No.12, 8422-8424, 1993.
141.
Takashi Murakami, Toshihiko Nishiyama, Tetsuya Shirotani, Yasuo Shinohara, Masaharu Kan, Kazuo Ishii, Fumihiko Kanai, Shoichi Nakazuru and Yousuke Ebina : Identification of two enhancer elements in the gene encoding the type 1 glucose transporter from the mouse which are responsive to serum, growth factor, and oncogenes., The Journal of Biological Chemistry, Vol.267, No.13, 9300-9306, 1992.
(Summary)
The type 1 glucose transporter (GLUT1) gene encodes an integral membrane glycoprotein responsible for facilitating transfer of glucose across plasma membrane and is rapidly activated by serum, growth factors, and by oncogenic transformation. To elucidate the molecular mechanisms of regulation of GLUT1 gene expression, we isolated and characterized the mouse GLUT1 gene. DNA elements regulating transcription of the gene were analyzed in transient expression assays after transfection of NIH/3T3 cells with a low background chloramphenicol acetyltransferase (CAT) vector system pSVOOCAT. We identified two enhancer elements; the first one is located 2.7 kilobases upstream of the cap site of the gene which contains the homologous sequences with two 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs), a serum response element, a cyclic AMP-responsive element (CRE) and three GC boxes, and the second one is located in the second intron of the gene which contains the homologous sequences with two TREs and one CRE. With the promoter alone the transcription of the gene is activated by src, only slightly activated by ras and is not activated by serum and platelet-derived growth factor. When the gene is accompanied by one of these enhancers, the transcription is activated by all these stimuli.
Eiichi Araki, Takashi Murakami, Tetsuya Shirotani, Fumihiko Kanai, Yasuo Shinohara, Fumio Shimada, Masataka Mori, Motoaki Shichiri and Yousuke Ebina : A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene., The Journal of Biological Chemistry, Vol.266, No.6, 3944-3948, 1991.
(Summary)
Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.
Yasuo Shinohara, Hideaki Nagamune and Hiroshi Terada : The hydrophobic cationic cyanine dye inhibits oxidative phosphorylation by inhibiting ADP transport, not by electrophoretic transfer, into mitochondria, Biochemical and Biophysical Research Communications, Vol.148, No.3, 1081-1086, 1987.
Eriko Obana, Masahiro Abe, Takenori Yamamoto and Yasuo Shinohara : The importance of hexokinase in the energy metabolism of tumor cells, Experimental Medicine, Vol.30, No.15, 36-41, Sep. 2012.
2.
Takenori Yamamoto, Akiko Yamada, Yuya Yoshimura, Hiroshi Terada and Yasuo Shinohara : The mechanisms of the release of cytochrome C from mitochondria revealed by proteomics analysis., Journal of the Pharmaceutical Society of Japan, Vol.132, No.10, 1099-1104, 2012.
(Summary)
Mitochondrial permeability transition (PT) is the phenomenon in which the mitochondrial inner membrane becomes permeable to various solutes and ions. When PT is induced by Ca(2+), cytochrome c is released from mitochondria into the cytosol where it then triggers subsequent steps of programmed cell death, apoptosis. Thus, the proteins that regulate PT and cytochrome c release could become druggable targets for various diseases. However, the mechanisms of PT and the release of cytochrome c have not yet been revealed. We previously showed that valinomycin, a potassium selective ionophore, also caused release of cytochrome c from mitochondria without inducing PT. This result indicates that cytochrome c could be released from mitochondria with or without induction of PT. In this study, to understand the difference of effects of valinomycin and Ca(2+) on mitochondria, we examined what protein species are released from valinomycin- and Ca(2+)-treated mitochondria by LC-MS/MS. As a result, only the proteins located in the intermembrane space were found to be released from valinomycin-treated mitochondria, while those in both the intermembrane space and in the matrix were released from Ca(2+)-treated mitochondria. Furthermore, the protein releases by each reagent occurred not selectively but in a concentration-dependent manner. Based on these results, the permeabilization effects of Ca(2+) and valinomycin on the inner and outer mitochondrial membranes are discussed.
Takenori Yamamoto, Akiko Yamada and Yasuo Shinohara : プロテオミクスで探るミトコンドリアからのシトクロムc漏出機構, Seibutsu Butsuri, Vol.50, No.3, 124-125, May 2010.
5.
Yasuo Shinohara, Mitsuru Hashimoto, Yoshitaka Kihira, Kazuto Ohkura, Eiji Majima and Hiroshi Terada : Structural and Functional Properties of the C-terminal Region of Mitochondrial ADP/ATP Carrier, Journal of the Pharmaceutical Society of Japan, Vol.130, No.2, 199-204, Feb. 2010.
(Summary)
Mitochondrial ADP/ATP carrier (AAC) is a protein catalyzing the transport of adenine nucleotides across inner mitochondrial membrane. In this review article, we first briefly introduce structural and functional properties of this protein. Next, we describe the results of our recent studies on the difference in the C-terminal region between yeast type 2 AAC isoform and bovine type 1 AAC isoform. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier are discussed.
(Keyword)
ADP/ATP carrier (AAC) / solute carrier family / mitochondria / transport mechanism / yeast / yeast
Masahiro Watanabe, Takenori Yamamoto, Chihiro Mori, Naoto Okada, Naoshi Yamazaki, Kazuaki Kajimoto, Masatoshi Kataoka and Yasuo Shinohara : Cold-induced changes in gene expression in brown adipose tissue: implications for the activation of thermogenesis., Biological & Pharmaceutical Bulletin, Vol.31, No.5, 775-784, May 2008.
(Summary)
Brown adipose tissue (BAT) is the site of heat production (thermogenesis). This unique function is performed by uncoupling protein 1 (UCP1) specifically expressed in mitochondria of BAT. UCP1 dissipates the driving force of ATP synthesis, and thus causes heat production followed by energy expenditure. The thermogenic function of BAT has the role of maintaining body temperature under cold conditions. When animals are exposed to cold, the expression of UCP1 gene is increased to activate thermogenesis. To date, functional analysis of BAT has been focused on UCP1, because it plays an indispensable role in thermogenesis. However, the gene expression of not only UCP1 but also that of other genes in BAT is expected to be regulated to achieve effective thermogenesis. Our previous investigations showed increased expression of genes that encode several energy metabolic enzymes in the BAT of rats kept in the cold. These changes in gene expression imply that the enhancement of energy metabolism is needed to activate thermogenesis. Furthermore, various reports from studies focused on genes whose expression is changed in response to cold stimulation have provided new insights into the function of BAT. In this review, to understand the thermogenic function of BAT systematically, we have provided an overview of previous findings on changes in the expression of genes thought to be related to the activation of thermogenesis in BAT.
(Keyword)
brown adipose tissue / Animals / Cold Temperature / gene expression / Humans / Thermogenesis
Masahiro Watanabe, Naoshi Yamazaki, Masatoshi Kataoka and Yasuo Shinohara : Exploratory Studies on Biomarkers : An Example Study on Brown Adipose Tissue, IEEJ Transactions on Electronics, Information and Systems, Vol.127, No.2, 198-203, Feb. 2007.
(Summary)
In mammals, two kinds of adipose tissue are known to exist, i.e., white (WAT) and brown (BAT) adipose tissue. The physiological role of WAT is storage of excess energy as fat, whereas that of BAT is the expenditure of excess energy as heat. The uncoupling protein UCP1, which is specifically expressed in brown fat mitochondria, dissipates the proton electrochemical potential across the inner mitochondrial membrane, known as a driving force of ATP synthesis, and thus it dissipates excess energy in a form of heat. Because deficiency in effective expenditure of excess energy causes accumulation of this energy in the form of fat (i.e., obesity), it is very important to understand the energy metabolism in this tissue for the development of anti-obesity drugs. In this article, in addition to providing a brief introduction to the functional properties of BAT and UCP1, the results of our exploratory studies on protein components involved in the energy-dissipating function in BAT.
(Keyword)
brown adipose tissue / energy metabolism / gene expression profiles / microarray
Drug-induced gingival overgrowth is a side effect associated with 3 types of drugs: anticonvulsants (phenytoin), immunosuppressive agents (cyclosporine A), and various calcium channel blockers for cardiovascular diseases. Gingival overgrowth is characterized by the accumulation of extracellular matrix in gingival connective tissues, particularly collagenous components with various degrees of inflammation. Although the mechanisms of these disorders have not been elucidated, recent studies suggest that these disorders seem to be induced by the disruption of homeostasis of collagen synthesis and degradation in gingival connective tissue, predominantly through the inhibition of collagen phagocytosis of gingival fibroblasts. The integrins are a large family of heterodimeric transmembrane receptors for extracellular matrix molecules. alpha2beta1 integrin serves as a specific receptor for type I collagen on fibroblasts, and alpha2 integrin has been shown to play a crucial role in collagen phagocytosis. Actin filaments, which are assembled from monomers and oligomers, are involved in collagen internalization after binding to integrins. Furthermore, the implication of intracellular calcium in the regulation of integrin-mediated binding activity and gelsolin activity, known as a calcium-dependent actin-severing protein, is also described. In this review, we focus on collagen metabolism in drug-induced gingival overgrowth, focusing on the regulation of collagen phagocytosis in fibroblasts.
(Keyword)
drug-induced gingival overgrowth / collagen phagocytosis / α2β1 integrin / type I collagen / fibroblast
Kazuaki Kajimoto, Naoshi Yamazaki, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Identification of possible protein machinery involved in the thermogenic function of brown adipose tissue., The Journal of Medical Investigation : JMI, Vol.51, No.1-2, 20-28, Feb. 2004.
(Summary)
Brown adipose tissue (BAT) is believed to function by dissipating excess energy in mammals. It is very important to understand the energy metabolism held in BAT since disorder of its energy-dissipating function may cause obesity or lifestyle-related diseases such as hypertension and diabetes. This function in BAT is mainly attributable to uncoupling protein (UCP), specifically expressed in its mitochondria. This protein consumes excess energy as heat by dissipating the H+ gradient across the inner mitochondrial membrane that is utilized as a driving force for ATP synthesis. In this review article, in addition to providing a brief introduction to the functional properties of BAT and UCP, we also describe and discuss properties of cultured brown adipocytes and the results of our exploratory studies on protein components involved in the energy-dissipating function in BAT.
Yasuo Shinohara, Takenori Yamamoto and 寺田 弘 : Recent Progress in Studies on The Uncouplers of Oxidative Phosphorylation., MEMBRANE, Vol.28, No.6, 271-277, Nov. 2003.
(Summary)
The chemical energy of nutrient molecules is converted in to ATP during the process of oxidative phosphorylation in mitochondria. Since the electrochemical potential gradient of H<SUP>+</SUP> formed across the inner mitochondrial membrane by the oxidation of respiratory substrates is utilized as a driving force for ATP synthesis, the mitochondrial inner membrane is highly impermeable to H<SUP>+</SUP>. An increase in the permeability of this membrane to H<SUP>+</SUP> causes inhibition of ATP synthesis, and chemicals that cause such an increase are called uncouplers. Ordinary known uncouplers are hydrophobic weak acids and show their activity by acting as protonophores. On the contrary, in recent studies, a variety of chemicals were found to act as uncouplers, not by acting as a protonophore, but by inducing permeability transition of the inner mitochondrial membrane or by inducing molecular ``slippage'' of the primary H<SUP>+</SUP> pump. In this review article, recent progress made from studies on the uncouplers of oxidative phosphorylation is presented and discussed.
Yasuo Shinohara, Kazuaki Kajimoto, Takiko Daikoku, Atsushi Shima, Naoshi Yamazaki and Hiroshi Terada : Bioenergetics in brown adipose tissue., Recent Res. Devel.Bioener., Vol.2, 1-14, 2002.
12.
Rieko Tachibana, Hideyoshi Harashima, Yasuo Shinohara and Hiroshi Kiwada : Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviarl vectors, Advanced Drug Delivery Reviews, Vol.52, No.3, 219-226, Nov. 2001.
13.
Hideyoshi Harashima, Yasuo Shinohara and Hiroshi Kiwada : Intracellular control of gene trafficking using liposomes as drug carriers, European Journal of Pharmaceutical Sciences, Vol.13, No.1, 85-89, Apr. 2001.
(Summary)
The objective of this review is to summarize some of the critical barriers in gene delivery and recent progress in overcoming such barriers using non-viral carrier systems. Receptor-mediated endocytosis is generally considered to be a principal entering pathway. Therefore, endosomal escape is an essential step for achieving efficient transfection. The nuclear membrane is also a critical barrier in gene delivery and the application of the nuclear localization signal is discussed, based on recent strategies. It is essential to optimize the carrier system, in order to enhance the transfection ability equivalent to a viral system. The importance of developing an intracellular pharmacokinetic model of genes is emphasized in the optimization of non-viral carrier systems.
(Keyword)
Liposomes / Drug delivery system / Gene delivery / Carrier system / Nuclear localization signal
Rieko Tachibana, Hideyoshi Harashima, Yasuo Shinohara and Hiroshi Kiwada : Intracellular control of macromolecules by use of liposomes as drug carriers, Journal of Biological Macromolecules, Vol.1, No.3, 60-67, 2001.
15.
Yasuo Shinohara : がん細胞の糖代謝を特異的に担うヘキソキナーゼの同定とその機能解析, Journal of the Pharmaceutical Society of Japan, Vol.120, No.8, 657-666, Aug. 2000.
(Keyword)
ヘキソキナーゼ / エネルギー代謝
16.
原島 秀吉, Yasuo Shinohara and Hiroshi Kiwada : キャリアによる細胞内デリバリー戦略と今後の課題, Drug Delivery System, Vol.14, 157-164, 1999.
Naoshi Yamazaki, Makiko Shinomiya, Hironobu Ike, Yasuo Shinohara, Noriaki Minakawa, Kouji Itou and Yoshiharu Takiguchi : Use of modified U1 small nuclear RNA for improved formation of properly spliced mRNA encoding human cathepsin A from the gene having an IVS7 +3a>g mutation, The 43rd FEBS Congress, Praha, Jul. 2018.
2.
Yuka Hiroshima, Eijiro Sakamoto, Kaori Abe, Kaya Yoshida, Koji Naruishi, Toshihiko Nagata, Yasuo Shinohara, Geczy Carolyn and Jun-ichi Kido : Advanced Glycation End-Products Increase Calprotectin in Human Gingival Epithelial Cells, The 95th General Session & Exhibition of the International Association for Dental Research (IADR), San Francisco, Mar. 2017.
3.
Naoshi Yamazaki, Yasuo Shinohara, Kouji Itou, Noriaki Minakawa and Yoshiharu Takiguchi : Rescue of mutation-induced exon 7 skipping in human Cathepsin A by using modified U1 small nuclear RNA, 2016 ASCB Annual Meeting, San Francisco, Dec. 2016.
4.
Yamagoshi Ryohei, Takenori Yamamoto, Mitsuru Hashimoto, Hiroshi Terada and Yasuo Shinohara : Functional expression of mammalian mitochondrial phosphate carrier in yeast cells, The American Society for Cell Biology 2016, 62, San Francisco, Dec. 2016.
5.
Takenori Yamamoto, Yamagoshi Ryohei, Harada Kazuki, Kawano Mayu, Minami Naoki, Ido Yusuke, Ozono Mizune, Watanabe Akira, Akiko Yamada, Hiroshi Terada and Yasuo Shinohara : Analysis of the Structure and Function of EMRE in Mitochondrial Calcium Channel using a Yeast Expression System, European Bioenergetics Conference 2016, Riva del Garda, Jul. 2016.
6.
Shima Sawako, Takenori Yamamoto, Yasuo Shinohara and Yoshiharu Takiguchi : Induction of mitochondrial permeability transition by dequalinium, 40th FEBS Congress, Berlin, Jul. 2015.
7.
Kuwahara Kana, Harada Kazuki, Yamagoshi Ryohei, Yoshiharu Takiguchi, Takenori Yamamoto and Yasuo Shinohara : Effects of employment of distinct strategies to capture antibody on antibody delivery into cultured cells, 40th FEBS Congress, Berlin, Jul. 2015.
8.
Ido Yusuke, Takenori Yamamoto, Yoshitomi Tatsuki and Yasuo Shinohara : Utility of syntenic relationships of VDAC1 pseudogenes for not only an understanding of the phylogenetic divergence history, but also ascertaining possible pseudogene candidates as genuine pseudogenes, The American Society for Cell Biology 2014, Philadelphia, Dec. 2014.
9.
Takenori Yamamoto, Ito Mika, Kageyama Keita, Kuwahara Kana, Kikuji Yamashita, Yoshiharu Takiguchi, Seiichiro Kitamura, Hiroshi Terada and Yasuo Shinohara : Mastoparan causes mitochondrial permeability transition not by interacting with specific proteins, but by interacting with the phospholipid phase, The American Society for Cell Biology 2014, Philadelphia, Dec. 2014.
10.
Ryohei Yamagoshi, Takenori Yamamoto and Yasuo Shinohara : Functional expression of the human mitochondrial phosphate carrier in yeast cells, The EMBO meeting 2013, A100, Amsterdam, Sep. 2013.
11.
Takuya Hada, Yumiko Kato, Eriko Obana, Naoshi Yamazaki, Takenori Yamamoto and Yasuo Shinohara : Comparison of two expression systems using COS7 Cells and yeast cells for expression of heart/muscle-type carnitine palmitoyltransferase 1, American Society for Cell Biology 2012 Annual Meeting, San Francisco, Dec. 2012.
12.
Yamamoto Atsushi, Takenori Yamamoto, Watanabe Masahiro, Hiroshi Terada and Yasuo Shinohara : Quantitative evaluation of the effects of cold exposure of rats on the expression levels of 10 FABP isoforms in brown adipose tissue, The Second Decennial Meeting between the University of Tokushima and Seoul National University on Pharmaceutical Sciences, Awaji, Dec. 2010.
13.
Yuka Nagasaki, S Yatsushiro, S. Yamamura, Kaori Abe, Yasuo Shinohara and Masatoshi Kataoka : High sensitive DNA detection with the combination of two kinds of fluorescent dyes by microchip electrophoresis, Pacifichem 2010, Honolulu, Dec. 2010.
14.
Taisuke Matsuo, Takenori Yamamoto, Katsuda Chie, Naoshi Yamazaki, Yasuo Shinohara and Masatoshi Kataoka : Substitution of Certain Amino Acids in a Short Peptide Causes a Significant Difference in Their Immunoreactivities with Antibodies Against Different Epitopes: Evidence for Possible Folding of the Peptide on Nitrocellulose or PVDF Membrane, International symposium on system cell engineering by multi-scale manipulation, Nagoya, Nov. 2008.
15.
Taisuke Matsuo, Naoshi Yamazaki, Tatsuhiro Ishida, Hiroshi Kiwada, Yasuo Shinohara and Masatoshi Kataoka : Design, preparation and directional insertion of peptides into lipid bilayer membrane and heir application for the preparation of liposome of which surface could be coated by externally added antibody, International symposium on system cell engineering by multi-scale manipulation, Nagoya, Nov. 2007.
16.
Taisuke Matsuo, Naoshi Yamazaki, Tatsuhiro Ishida, Hiroshi Kiwada, Yasuo Shinohara and Masatoshi Kataoka : Mutant coat proteins of Pf3 bacteriophage as models of membrane proteins and their interactions with lipid bilayer membrane, Pre-Satellite Meeting of the 3rd Pharmaceutical Sciences World Congress (PSWC 2007) for and by Ph.D. students and postdoctoral fellows, Amsterdam, Apr. 2007.
17.
Akihiro Iwahasi, Naoshi Yamazaki, Hiroshi Terada and Yasuo Shinohara : Structural analysis of C-terminal region of mitochondrial ADP/ATP carrier by site-directed mutagenesis and chemical modifications, Pre-Satellite Meeting of the 3rd Pharmaceutical Sciences World Congress (PSWC 2007) for and by Ph.D. students and postdoctoral fellows, Amsterdam, Apr. 2007.
18.
Masahiro Watanabe, Takenori Yamamoto, Rei Kakuhata, Kazuaki Kajimoto, Fumiyo Kita, Naoshi Yamazaki, Yoshinobu Baba and Yasuo Shinohara : Synchronized changes in the transcript levels of the genes towards activated thermogenesis in brown adipose tissue induced by cold exposure of experimental animals, Pre-Satellite Meeting of the 3rd Pharmaceutical Sciences World Congress (PSWC 2007) for and by Ph.D. students and postdoctoral fellows, Amsterdam, Apr. 2007.
19.
Rei Kakuhata, Takenori Yamamoto, Masahiro Watanabe, Naoshi Yamazaki, Masatoshi Kataoka, Yoshinobu Baba and Yasuo Shinohara : Possible utilization of synthetic mRNAs as standards for microarray analysis, Pre-Satellite Meeting of the 3rd Pharmaceutical Sciences World Congress (PSWC 2007) for and by Ph.D. students and postdoctoral fellows, Amsterdam, Apr. 2007.
20.
Toshihiko Ooie, Masato Tanaka, Tomonori Nakahara, Shinji Hyodo, Yasuo Shinohara, Hidetoshi Miyashita and Yoshinobu Baba : Laser-controlled pico-injector for nano-biodevices, Proceedings of Laser Precision Microfabrication 2007 LPM2007, Wien, Apr. 2007.
21.
Taisuke Matsuo, Naoshi Yamazaki, Tatsuhiro Ishida, Hiroshi Kiwada, Yasuo Shinohara and Masatoshi Kataoka : Mutant coat proteins of Pf3 bacteriophage as models of membrane proteins and their interactions with lipid bilayer membrane, International symposium on system cell engineering by multi-scale manipulation, Nagoya, Nov. 2006.
22.
Akihiro Iwahashi, Yoshitaka Kihira, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Dynamic conformational change of fifth transmembrane segment of yeast type 2 ADP/ATP carrier is involved in its nucleotide exchange function, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jun. 2006.
23.
Naoshi Yamazaki, Saki Imamura, Hirokazu Sato, Arisa Hironaga, Takenori Yamamoto, Yasuo Shinohara and Hiroshi Terada : cDNA from Rat Brown Adipose Tissue Codes a Novel Protein Containing DH, BAR, and SH3 Domains, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jun. 2006.
24.
Takenori Yamamoto, Naoshi Yamazaki, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Two critical factors affecting the release of mitochondrial cytochrome c as revealed by studies using N,N'-dicyclohexylcarbodiimide as an inducer of permeability transition, The 11th Korea-Japan Joint Symposium on Drug Design and Development, Cheju, May 2006.
25.
Toshihiko Ooie, Masato Tanaka, Tomonori Nakahara, Shinji Hyodo, Yasuo Shinohara and Yoshinobu Baba : Laser Controlled Pico-Fraction Unit for Nano-biodevices, Proc. of MicroTAS2006, Tokyo, 2006.
26.
Takenori Yamamoto, Satsuki Terauchi, Aiko Tachikawa, Kikuji Yamashita, Masatoshi Kataoka, Hiroshi Terada and Yasuo Shinohara : Two Critical Factors Affecting the Release of Mitochondrial Cytochrome c as Revealed by Studies Using N,N'-Dicyclohexylcarbodiimide as an Inducer of Permeability Transition, 45th American Society for Cell Biology Annual Meeting, San Francisco, Dec. 2005.
27.
Akiko Yamada, Takenori Yamamoto, Masatoshi Kataoka, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara : Induction of Permeability Transition in Yeast Mitochondria Causes Release of Mitochondrial Cytochrome c, 45th American Society for Cell Biology Annual Meeting, San Francisco, Dec. 2005.
28.
Akihiro Iwahashi, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Structural Change of the Cytosolic Second Loop of Mitochondrial ADP/ATP Carrier Revealed by Cysteine-scanning Mutagenesis, 45th American Society for Cell Biology Annual Meeting, San Francisco, Dec. 2005.
29.
Yasuo Shinohara : Strategies used for functional characterization of membrane proteins, International Workshop on Surface-Biotronics, 5-6, Tokyo, Oct. 2004.
30.
Yoshitaka Kihira, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Twist of second transmembrane alpha-helix of mitochondrial ADP/ATP carrier, Pharmaceutical sciences world congress(PSWC2004), Kyoto, Jun. 2004.
31.
Takenori Yamamoto, Satsuki Terauchi, Hiroshi Terada and Yasuo Shinohara : DCCD induces mitochondrial permeability transition without causing cytochrome c release, Pharmaceutical sciences world congress(PSWC2004), Kyoto, Jun. 2004.
32.
Yoshitaka Kihira, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Twist of second transmembrane alpha-helix of mitochondrial ADP/ATP carrier, Pharmaceutical sciences world congress(PSWC2004), Kyoto, Jun. 2004.
33.
Kazuaki Kajimoto, Hiroshi Terada and Yasuo Shinohara : The role of citrate transport from mitochondria in the regulation of fat accumulation, Pharmaceutical sciences world congress(PSWC2004), Kyoto, Jun. 2004.
34.
Yoko Fukura, Masatoshi Kataoka, Yasuo Shinohara and Yoshinobu Baba : Measurement of mitochondrial membrane potential on a microfabricated chip, Pharmaceutical sciences world congress(PSWC2004), Kyoto, Jun. 2004.
35.
Sonoko Inoue, Masatoshi Kataoka, Yasuo Shinohara and Yoshinobu Baba : Usefulness of microchip electrophoresis for reliable analyses of non-standard DNA samples and subsequent on-chip enzymatic digestion, Pharmaceutical sciences world congress(PSWC2004), Kyoto, Jun. 2004.
36.
Yasuo Shinohara : Identification of possible protein machinery involved in the thermogenic function of brown adipose tissue, The 10th Japan-Korea Joint symposium on drug design and development, Tokushima, Apr. 2004.
37.
Yasuo Shinohara, MR Almofti, Taro Ishida and Hiroshi Terada : Valinomycin Induces Permeability Transition Independent Release of Mitochondrial Cytochrome C, 42nd American Society for Cell Biology Annual Meeting, San Francisco, Dec. 2002.
38.
Mami Hino, Yasuo Shinohara and Hiroshi Terada : Effect of Serum on Glucose transporter and Hexokinase in Fibroblast 3Y1, 42nd American Society for Cell Biology Annual Meeting, San Francisco, Dec. 2002.
39.
Almofti MR., Yasuo Shinohara and Hiroshi Terada : Novel Pore Transiently Opened in the Inner Mitochondrial Membrane by Silver Ions is Associated with Release of Appotogenic Cytochrome C, 42nd American Society for Cell Biology Annual Meeting, San Francisco, Dec. 2002.
40.
Kazuaki Kajimoto, Takiko Daikoku, Naoshi Yamazaki, Yasuo Shinohara and Hiroshi Terada : Dominant Expression of NAD+-dependent Isocitrate Dehydrogenase in Brown Adipose Tissue, 42nd American Society for Cell Biology Annual Meeting, San Francisco, Dec. 2002.
41.
Naoshi Yamazaki, Yasuo Shinohara and Hiroshi Terada : High Expression of a Novel Carnitine Palmitoyltransferase I in Brown Adipose Tissue and Muscle: Isolation and Characterization of its cDNA and Genomic Clones, The 9th Korea-Japan Joint Symposium on Drug Design and Development, Seoul, May 2002.
42.
Yasuo Shinohara, Ishida Taro and Hiroshi Terada : Why do mitochondria from tumor cells show higher capability for hexokinase binding than those from normal cells ?, 3rd Albany conference on frontiers of mitochondrial research, Rensselaerville, NY, Sep. 2000.
43.
Yasuo Shinohara : Synchronized elevation of transcript levels of type 1 glucose transporter and type II hexokinase in tumor cells, 25 th Silver jubilee FEBS meeting, Copenhagen, Jul. 1998.
44.
Naoshi Yamazaki, Yasuo Shinohara and Hiroshi Terada : Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I, 12th International Symposium of Federation of Asian and Oceanian Biochemists and Molecular Biologist, Tokushima, Jul. 1996.
Proceeding of Domestic Conference:
1.
Takeshi Ito, Sayaka Kajita, FUJII Minori and Yasuo Shinohara : Heterologous expression and function of Plasmodium parasite malate-quinone oxidoreductase in yeast mitochondria, The 2023 Annual Conference of the Japan Society for Bioscience, Biotechnology and Agrochemistry, Mar. 2023.
2.
Takeshi Ito, Sayaka Kajita, FUJII Minori and Yasuo Shinohara : Plasmodium parasite malate-quinone oxidoreductase can be functionally expressed in yeast mitochondria, The 48rd Annual Symposium of The Japan Bioenergetics Group, Dec. 2022.
Kana Kuwahara, Kazuki Harada, Ryohei Yamagoshi, Takenori Yamamoto and Yasuo Shinohara : Effects of the employment of distinct strategies to capture antibody against the antibody delivery into cultured cells, 日本薬学会年会, Mar. 2015.
41.
Ryohei Yamagoshi, Takenori Yamamoto and Yasuo Shinohara : Functional expression of the mammalian mitochondrial phosphate carrier in yeast mitochondria achieved by deletion presequence and mutation of the specific amino acid residues, 日本薬学会年会, Mar. 2015.
Kuwahara Kana, Harada Kazuki, Takenori Yamamoto and Yasuo Shinohara : Comparison of the delivery effects of two antibody carrier systems, 第52回日本生物物理学会年会, s106, Sep. 2014.
45.
Kotake Kazumasa, Inotani Yuki, Yoshimura Yuya, Harada Kazuki, Takenori Yamamoto and Yasuo Shinohara : Synergistic inhibitory effects of oligomycin and rotenone on the mitochondrial permeability transition, 第52回日本生物物理学会年会, s107, Sep. 2014.
46.
Ryouhei Yamagoshi, Takenori Yamamoto and Yasuo Shinohara : 哺乳類のリン酸輸送担体を酵母のミトコンドリアに機能的に発現させるために必要な因子の理解に向けて, 第6回日本生物物理学会第6回中国四国支部大会, 32, May 2014.
47.
Tatsuki Yoshitomi, Ryohei Yamagoshi, Takenori Yamamoto and Yasuo Shinohara : Functional expression of human mitochondrial S-adenosylmethionine carrier in yeast., 日本薬学会第134年会, Mar. 2014.
48.
Yuusuke Ido, Tatsuki Yoshitomi, Kazuto Ohkura, Takenori Yamamoto and Yasuo Shinohara : 3つのVDAC1の偽遺伝子はラットとマウスの分岐前に形成されていた, 日本薬学会第134年会, Mar. 2014.
49.
Keita Kageyama, Yasuo Shinohara, Takenori Yamamoto, Naoshi Yamazaki and Yoshiharu Takiguchi : ミトコンドリア透過性遷移におけるシクロフィリンDの挙動解析, 第87回日本薬理学会年会, Mar. 2014.
秦 拓也, 加藤 弓子, Eriko Obana, Naoshi Yamazaki, Takenori Yamamoto and Yasuo Shinohara : Comparison of two expression systems using COS7 cells and yeast Cells for expression of heart/muscle-type CPT1, 第50回日本生物物理学会, Sep. 2012.
66.
Takenori Yamamoto, Yusuke Ido, Yumiko Nakano, Mayu Kawano, Hiroshi Terada, Hideyoshi Harashima and Yasuo Shinohara : 抗原抗体反応とプロテオミクスを用いたミトコンドリア外膜に存在するVDACアイソフォームの発現プロファイル解析, 日本DDS学会, Jul. 2012.
Eriko Obana, Takuya Hada, Takenori Yamamoto, Tomosige Hori and Yasuo Shinohara : Affymetrix社のGeneChip Rat Gene 1.0 ST Arrayにおいて観察される各プローブのシグナル強度の解析, 日本薬学会第132年会, Mar. 2012.
72.
Takenori Yamamoto, Akiko Yamada, Yuya Yoshimura, Hiroshi Terada and Yasuo Shinohara : プロテオミクスで解き明かすミトコンドリアからのシトクロムc放出機構, 日本薬学会第132年会, Mar. 2012.
Yasuo Shinohara, 橋本 満, Yoshitaka Kihira, 大倉 一人, Eiji Majima and Hiroshi Terada : Structural and functional properties of the C-terminal region of mitochondrial ADP/ATP carrier, 日本薬学会 第129年会, Mar. 2009.
Naoshi Yamazaki, Hirokazu Sato and Yasuo Shinohara : Characterization of a Novel Protein from Rat Brown Adipose Tissue, Containing DH, BAR, and SH3 Domains., 第30回日本分子生物学会・第80回日本生化学会合同大会, Dec. 2007.
岩橋 晶洋, Naoshi Yamazaki and Yasuo Shinohara : Structural analysis of C-terminal region of mitochondrial ADP/ATP carrier by site-directed mutagenesis, 日本分子生物学会フォーラム, Dec. 2006.
112.
Akiko Yamada, Takenori Yamamoto, Naoshi Yamazaki, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara : Differential protein release from mitochondria induced by Ca2+ and valinomycin as revealed by immunological and proteomics techniques, 第6回ミトコンドリア学会, Dec. 2006.
113.
Takenori Yamamoto, Akiko Yamada, masahiro watanabe, yuya yoshimura, Naoshi Yamazaki, Yoshiyuki Yoshimura, Takashi Yamauchi, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara : Expression levels of voltage-dependent anion channel (VDAC) isoforms in mitochondrial outer membrane revealed by immunological and proteomics techniques, 第28回生体膜と薬物の相互作用シンポジウム, Nov. 2006.
114.
前田 瑛起, Masatoshi Kataoka, Yoshinobu Baba and Yasuo Shinohara : マイクロチップを用いた血中グルコース濃度測定法の開発, 第45回 日本薬学会・日本病院薬剤師会 中国四国支部学術大会, Oct. 2006.
Naoshi Yamazaki, saki imamura, hirokazu sato, Takenori Yamamoto, Yasuo Shinohara and Hiroshi Terada : Characterization of Novel cDNA from Rat Brown Adipose Tissue: It Codes a Protein Containing Dbl Homology, Bin/Amphiphysin/Rvs and Src Homology 3 Domains, 第78回日本生化学会大会, Oct. 2005.
岩橋 晶洋, Yoshitaka Kihira, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Structural characterization of the cytosolic second loop of mitochondrial ADP/ATP carrier by site directed mutagenesis, 第77回 日本生化学会大会, Oct. 2004.
喜多 史代, 梶本 和昭, Hiroshi Terada and Yasuo Shinohara : UCP1の機能を制御するプリンヌクレオチドの定量的評価, 日本薬学会第124年会要旨集, Mar. 2004.
137.
梶本 和昭, Hiroshi Terada and Yasuo Shinohara : 脂肪蓄積の制御におけるミトコンドリアからのクエン酸輸送の役割, 日本薬学会第124年会要旨集, Mar. 2004.
138.
Yoshitaka Kihira, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Structural analysis of cytosoloc first loop of mitochondrial ADP/ATP carrier by using point mutations and chemical modification, 第25回 生体膜と薬物相互作用シンポジウム, Nov. 2003.
139.
木平 孝高, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Structural analysis of cytosoloc first loop of mitochondrial ADP/ATP carrier by using point mutations and chemical modification, 第25回 生体膜と薬物相互作用シンポジウム, Nov. 2003.
140.
Yoshitaka Kihira, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Detection of conformational change of the cytosolic first loop of mitochondrial ADP/ATP carrier using SH reagent, 第76回 日本生化学会大会, Oct. 2003.
141.
立川 愛子, 市川 智一, 中川 良典, Hiroshi Terada and Yasuo Shinohara : Multiple effects of cyanine dye on the structure and function of mitochondria, 第76回 日本生化学会大会, Oct. 2003.
142.
寺内 さつき, Takenori Yamamoto, Kikuji Yamashita, Hiroshi Terada and Yasuo Shinohara : DCCD induces mitochondrial permeability transition without causing cytochrome c release, 第76回 日本生化学会大会, Oct. 2003.
143.
木平 孝高, Eiji Majima, Hiroshi Terada and Yasuo Shinohara : Detection of conformational change of the cytosolic first loop of mitochondrial ADP/ATP carrier using SH reagent, 第76回 日本生化学会大会, Oct. 2003.
144.
久保井 覚, 鶴本 智大, 佐川 幾子, Eiji Majima, Yasuo Shinohara and Hiroshi Terada : がん細胞ミトコンドリアにおけるADP/ATP透過担体複合体のアイソフォーム変動, 日本薬学会 第123年会, Mar. 2003.
145.
Yoshitaka Kihira, 畑中 貴, Eiji Majima, Yasuo Shinohara and Hiroshi Terada : ADP/ATP透過担体二量体における各分子の異なる役割, 日本薬学会 第123年会, Mar. 2003.
三木 里美, 畑中 貴, Satoru GOTO, 真島 英司, Yasuo Shinohara and 寺田 弘 : ミトコンドリアADP/ATP透過担体の輸送活性発現におけるArg151とAsp167の役割, 日本薬学会第121年会, Mar. 2001.
162.
Yoshitaka Kihira, 畑中 貴, Eiji Majima, Yasuo Shinohara and Hiroshi Terada : Characterization of the cytosol-oriented regions of the mitochondrial ADP/ATP carrier by site-directed chemical modification, 第74回 日本生化学会大会, 2001.
163.
Naoshi Yamazaki, 新藤 雅之, Yasuo Shinohara and Hiroshi Terada : ヒト筋型CPTIとCK/EKの2つの遺伝子領域を含んだmRNAのキャラクタリゼーション, 日本薬学会 第120年会, Mar. 2000.
Naoshi Yamazaki, 島 厚志, Yasuo Shinohara and Hiroshi Terada : 褐色脂肪組織に特異的なタンパク質をコードするcDNAの単離と構造解析, 日本薬学会 第115年会, Mar. 1995.
181.
Naoshi Yamazaki, Yasuo Shinohara and Hiroshi Terada : 褐色脂肪組織に特異的なタンパク質をコードする遺伝子の解析, 日本薬学会 114年会, Mar. 1994.
182.
紙田 真喜夫, Naoshi Yamazaki, Yasuo Shinohara and Hiroshi Terada : ラットミトコンドリアADP/ATP透過担体をコードする遺伝子の転写調節機構の解析, 日本薬学会 第112年会, Mar. 1992.
183.
小池 晴彦, Yasuo Shinohara, Satoru GOTO, Hiroshi Terada and Zenei Taira : Monte Carlo法を用いた薬物の動的構造の評価と構造活性相関, 第16回構造活性相関シンポジウム, Nov. 1988.
Et cetera, Workshop:
1.
Naoshi Yamazaki, Makiko Shinomiya, Hironobu Ike, Yasuo Shinohara, Noriaki Minakawa, Kouji Itou and Yoshiharu Takiguchi : Use of modified U1 small nuclear RNA for improved formation of properly spliced mRNA encoding human cathepsin A from the gene having an IVS7 +3a>g mutation, FEBS Open Bio, Vol.8, No.Supplement 1, ShT.35-1, Jul. 2018.
梶本 和昭, Naoshi Yamazaki, 島 厚志, Hiroshi Terada, Yasuo Shinohara and Yoshinobu Baba : 褐色脂肪組織におけるエネルギー代謝系のキャラクタリゼーション, 平成14年度産業技術総合研究所四国センターシンポジウム, Dec. 2002.
6.
Naoshi Yamazaki, Yasuo Shinohara and Hiroshi Terada : Novel carnitine palmitoyltransferase I isoform in rat BAT, The Joint Symposium between Seoul National University and University of Tokushima on Pharmacy (10th Anniversary of Exchange Program), Aug. 2000.
Validation of the usefulness of the method for elucidating the interaction between membrane proteins and ligands based on the acquisition of highly efficient revertant strains (Project/Area Number: 20K05750 )
Use of highly efficient procedure in getting revertant cells, for studies of interaction manner between membrane protein and its inhibitor (Project/Area Number: 17K08274 )
Basic study for oral care system using an artificial cell (Project/Area Number: 17H04418 )
Studies on the mitochondrial proteins responsible for the traffic control across mitochondrial outer membrane, and its application for drug design (Project/Area Number: 25460066 )
Molecular mechanisms of permeability transition revealed using yeast mitochondria (Project/Area Number: 22790070 )
Bioactive compounds utilizing interactions between cell membrane and membrane proteins (Project/Area Number: 22659023 )
In vivo induction of brown ad ipocyte differentiation by noninvasive delivery of miRNAs into stem cell (Project/Area Number: 20200064 )
Research on hypoglycemia caused by inhibition of orexin expression (Project/Area Number: 18590991 )
Characterization of cell-free protein synthesis system and theapplication for the synthesis of cell membrane protein (Project/Area Number: 17076011 )
Mechanism of encephalitis after influenza virus infection based on defect of β-oxidation in liver (Project/Area Number: 15590942 )
Pulmonary delivery of microspheres loaded with antituberculosis agents to alveolar macrophages for development of antituberculosis therapy (Project/Area Number: 15300170 )
Optimization of drug delivery system for antitumor agents based on the energy metabolism inhibition of tumor cells (Project/Area Number: 09557197 )
Design and development of anti-tumor drugs using the energy metabolism specifically held in tumor cells as potential targets (Project/Area Number: 09357020 )
Studies on the specific pathway of energy metabolism held in tumor cells and their application on the development of anti-tumor drugs (Project/Area Number: 07557164 )