Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
34 巻 , 1 号
選択された号の論文の32件中1~32を表示しています
Analytical Biochemistry
Note
  • Naoki Tanaka, Keiko Nagasaka, Yasuhiko Komatsu
    原稿種別: Note
    専門分野: Analytical Biochemistry
    2011 年 34 巻 1 号 p. 135-141
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Selected reaction monitoring (SRM) mass spectrometry (MS) is becoming a popular approach for targeted quantitative proteomics. Triple-quadrupole mass spectrometers have been historically considered as the instrument of choice for this type of quantitative analysis. Recently, however, it has been reported that the SRM MS with a linear ion-trap (LIT) mass spectrometer is rather more appropriate for quantitative analysis of large peptides than the triple-quadrupole ones. In this study, we demonstrate that the SRM MS performed with a LIT mass spectrometer can simultaneously analyze multiple peptides and can quantify specific peptides in biological specimens without the use of stable isotope (SI)-labeled standard peptides. Firstly, a mixture of 10 synthesized peptides derived from yeast proteins and bovine serum albumin (BSA) was simultaneously analyzed by the LIT SRM. The ion peak areas of the 10 peptides were linearly correlated with the input amounts between 1 fmol and 10 pmol. Furthermore, the same peptide mixture spiked into human plasma was analyzed, and a linear response was found. Next, the amount of a BSA tryptic peptide was quantified by using an SI-labeled or a non SI-labeled peptide as an external reference standard. The difference in the quantified amounts of the BSA tryptic peptide was less than 10% between the 2 methods, suggesting that the “externally pulsed” non SI-labeled standard peptides derived from another species are useful. These results indicate that the SRM MS conducted with a LIT mass spectrometer is applicable to targeted quantitative proteomics of peptides at least up to 10 in number.
Biochemistry
Regular Articles
  • Yusuke Iguchi, Tomoko Nishimaki-Mogami, Masafumi Yamaguchi, Fumiteru T ...
    原稿種別: Regular Article
    専門分野: Biochemistry
    2011 年 34 巻 1 号 p. 1-7
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    The aim of this study is to examine the ability of the bile acid analogues obtained by chemical modification of ursodeoxycholic acid (UDCA) for TGR5 activation. Eleven UDCA analogues including 3- or 7-methylated UDCAs and amino acid conjugates were investigated as to their ability to activate TGR5 by means of the luciferase assay. It was noteworthy that 7α-methylated UDCA, namely 3α,7β-dihydroxy-7α-methyl-5β-cholanoic acid, had a significantly high affinity for and ability to activate TGR5 as compared to UDCA. Additionally, FXR activation ability of 7α-methylated UDCA was low relative to that of UDCA. However, other modification of UDCA, such as the introduction of methyl group at its C-3 position and oxidation or epimerization of hydroxyl group in the C-3 position, could not elicit such remarkable effect. The present findings would provide a useful strategy for the development of TGR5-selective agonist.
  • Koji Higai, Chiho Suzuki, Yuzo Imaizumi, Xin Xin, Yutaro Azuma, Kojiro ...
    原稿種別: Regular Article
    専門分野: Biochemistry
    2011 年 34 巻 1 号 p. 8-12
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Lectin-like receptors natural killer group 2D (NKG2D) and CD94 on natural killer (NK) cells bind to α2,3-NeuAc-containing N-glycans and heparin/heparan sulfate (HS). Using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rGST-NKG2Dlec) and CD94 (rGST-CD94lec), we evaluated their binding affinities (Kd) to high sialyl Lewis X (sLeX)-expressing transferrin secreted by HepG2 cells (HepTf) and heparin-conjugated bovine serum albumin (Heparin-BSA), using quartz crystal microbalance (QCM) and enzyme immunoassay (EIA) microplate methods. Kd values obtained by linear reciprocal plots revealed good coincidence between the two methods. Kd values of rGST-NKG2Dlec obtained by QCM and EIA, respectively, were 1.19 and 1.11 μM for heparin-BSA >0.30 and 0.20 μM for HepTf, while those of rGST-CD94lec were 1.31 and 1.45 μM for HepTf >0.37 and 0.36 μM for heparin-BSA. These results suggested that these glycans can interact with NKG2D and CD94 to modulate NK cell-dependent cytotoxicity.
  • Yuko Ogawa, Yuri Miura, Akira Harazono, Masami Kanai-Azuma, Yoshihiro ...
    原稿種別: Regular Article
    専門分野: Biochemistry
    2011 年 34 巻 1 号 p. 13-23
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30—100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.
  • Yoshikuni Goto, Rina Yoshioka, Naomi Arisaka, Akira Hattori, Masafumi ...
    原稿種別: Regular Article
    専門分野: Biochemistry
    2011 年 34 巻 1 号 p. 24-27
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Human laeverin/aminopeptidase Q (APQ) is a novel member of the M1 family of zinc aminopeptidases and is specifically expressed on the cell surface of extravillous trophoblasts. In this study, we examined the significance of Gln-238 of laeverin/APQ, a putative S1 site residue, by site-directed mutagenesis for its enzymatic activity and substrate specificity. Replacement of Gln-238 with Ala caused a significant change in substrate specificity rather than a decrease in enzymatic activity. These results indicate that Gln-238 is important for the substrate specificity of laeverin/APQ. In addition, our data suggest that direct electrostatic interaction between substrate and S1 site of the enzyme is not involved in the mutant enzyme's preference for basic amino acids.
Molecular and Cell Biology
Regular Articles
  • FuJun Liu, ShaoHua Jin, Ning Li, Xin Liu, HaiYan Wang, JianYuan Li
    原稿種別: Regular Article
    専門分野: Molecular and Cell Biology
    2011 年 34 巻 1 号 p. 28-35
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    The testis is the special male gonad responsible for spermatogenesis and steroidogenesis with complex gene expressions. Characterizing and comparing the testis-specific genes in different species can reveal key genes related to testis specifical functions and provide supplementary information for study of human testis function. We screened testis-specific genes from Unigene libraries, total 317, 449 and 147 testis-specific genes were identified for human, mouse and rat, respectively. Ten from thirteen selected human testis-specific genes were validated exclusively expressed in the testis by reverse transcription polymerase chain reaction (RT-PCR). Systematic bioinformatics analysis showed that specific genes were mainly related to spermatogenesis and testis development process with significant Glycolysis and Pyruvate metabolism. Enrichment functions were discussed.
  • Yasuyuki Fujimoto, Aya Kamakura, Yu Motohashi, Ayako Ohashi-Kobayashi, ...
    原稿種別: Regular Article
    専門分野: Molecular and Cell Biology
    2011 年 34 巻 1 号 p. 36-40
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    The carboxyl terminus of a human ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL), was tagged with green fluorescence protein (GFP), and the resulting fusion protein (TAPL-GFP) was stably expressed in Chinese hamster ovary (CHO)-K1 cells. The GFP signal was co-localized with that of LysoTracker but not that of MitoTracker, as visualized under a microscope. TAPL-GFP was co-sedimented with lysosomal marker cathepsin D on Percoll density gradient centrifugation. These results indicated that TAPL is a lysosomal ABC transporter but not a mitochondrial one. It was not solubilized completely with a non-ionic detergent under ice-cold conditions, and was co-sedimented with flotillin-1 on sucrose density gradient centrifugation. A similar result was obtained with high pH-treatment. Furthermore, treatment with methyl-β-cyclodextrin resulted in an altered distribution of TAPL-GFP. These results suggest that TAPL may be localized to the microdomains (lipid rafts) of lysosomal membranes enriched in cholesterol.
  • Eun Jeong Choo, Yun-Hee Rhee, Soo-Jin Jeong, Hyo-Jung Lee, Hyun Seok K ...
    原稿種別: Regular Article
    専門分野: Molecular and Cell Biology
    2011 年 34 巻 1 号 p. 41-46
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Anethole is known to possess anti-inflammatory and anti-tumor activities and to be a main constituent of fennel, anise, and camphor. In the present study, we evaluated anti-metastatic and apoptotic effects of anethole on highly-metastatic HT-1080 human fibrosarcoma tumor cells. Despite weak cytotoxicity against HT-1080 cells, anethole inhibited the adhesion to Matrigel and invasion of HT-1080 cells in a dose-dependent manner. Anethole was also able to down-regulate the expression of matrix metalloproteinase (MMP)-2 and -9 and up-regulate the gene expression of tissue inhibitor of metalloproteinase (TIMP)-1. The similar inhibitory effect of anethole on MMP-2 and -9 activities was confirmed by zymography assay. Furthermore, anethole significantly decreased mRNA expression of urokinase plasminogen activator (uPA), but not uPA receptor (uPAR). In addition, anethole suppressed the phosphorylation of AKT, extracellular signal-regulated kinase (ERK), p38 and nuclear transcription factor kappa B (NF-κB) in HT-1080 cells. Taken together, our findings indicate that anethole is a potent anti-metastatic drug that functions through inhibiting MMP-2/9 and AKT/mitogen-activated protein kinase (MAPK)/NF-κB signal transducers.
  • Wen-Jun Duan, Qi-Sheng Li, Ming-Yu Xia, Shin-Ichi Tashiro, Satoshi Ono ...
    原稿種別: Regular Article
    専門分野: Molecular and Cell Biology
    2011 年 34 巻 1 号 p. 47-53
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H2O2), superoxide dismutase (SOD) (scavenger of O2•−), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H2O2 or O2•− showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.
  • Toshiyuki Oshima, Tomofumi Fujino, Ken Ando, Makio Hayakawa
    原稿種別: Regular Article
    専門分野: Molecular and Cell Biology
    2011 年 34 巻 1 号 p. 54-60
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    FGD1 encodes a guanine nucleotide exchange factor for Cdc42. Mutations in the FGD1 gene are responsible for an X-linked disorder known as Aarskog-Scott syndrome (AAS). While most mutations were found in the catalytic region, which consists of Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, a missense mutation in the proline-rich domain is also found in a patient with typical clinical features as AAS. In this mutant FGD1, the serine residue at 205 is replaced with isoleucine. We recently demonstrated that FGD1 translocated to the membrane in response to extracellular stimuli such as epidermal growth factor (EGF) whereas FGD1 with S205/I substitution did not. Here we show that the proline-rich domain is critical for FGD1-induced directionally persistent cell migration. When inducibly expressed in HeLa Tet-Off cells, FGD1 stimulates directional migration whereas FGD1 with S205/I substitution does not affect it. We further demonstrate that FGD1 augments EGF-stimulated c-Jun NH2-terminal kinase (JNK) activation. In the presence of JNK inhibitor SP600125, motility of FGD1-expressing cells is significantly impaired, indicating a critical role of JNK in cell migration. However, FGD3, an FGD1 homologue lacking the proline-rich domain, and FGD1 with S205/I substitution augment EGF-stimulated JNK activation similarly to FGD1, suggesting that the proline-rich domain is not involved in the regulation of JNK. Finally, we show that FGD1, but not FGD1 with S205/I substitution, is phosphorylated in response to EGF, suggesting that the phosphorylation of S205 may trigger the FGD1 translocation to the leading edge membrane and enable cells to undergo directional migration.
Notes
  • Atsuko Matsumoto, Ken-ichiro Matsumoto, Shingo Matsumoto, Fuminori Hyo ...
    原稿種別: Note
    専門分野: Molecular and Cell Biology
    2011 年 34 巻 1 号 p. 142-145
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO2) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO2 in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO2 were compared. The pO2 in the blood can depend on the oxygen content in the breathing gas while tissue pO2 was not directly influenced by pO2 in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO2 is similar to normal tissue in the peripheral region.
  • Kazunori Saeki, Mayumi Machida, Yutaro Kinoshita, Ryoko Takasawa, Sei- ...
    原稿種別: Note
    専門分野: Molecular and Cell Biology
    2011 年 34 巻 1 号 p. 146-149
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase that phosphorylate protein substrates involved in Alzheimer's disease (AD), such as microtubule-associated protein tau and amyloid precursor protein (APP). GSK-3β consists of two splice variants; the major short form (GSK-3β1) distributes in many organs and the minor long form (GSK-3β2), whose structural difference is the insert of only 13 amino acid residues to the C-terminal side of the catalytic site of GSK-3β1, is present in central nervous system. However, the physiological significances of the two variants are unclear. Here we examined whether the phosphorylation activities of two variants to tau and APP are different in cells. We found that GSK-3β2 has lower phosphorylation activity to tau at AD-relevant epitope (Ser396) than GSK-3β1 in cells, whereas the two variants exhibit equivalent levels of phosphorylation activities to APP. Recombinant GSK-3β2 has also lower phosphorylation activity to tau than GSK-3β1 in vitro, although the phosphorylation activities of the two variants to a synthetic peptide substrate pGS-2 are comparable. Furthermore, the deletion of the C-terminal tail (CT) of GSK-3β2 resulted in considerable reduction of tau phosphorylation activity as compared with GSK-3β1, suggesting that the lower phospho-rylation activity of GSK-3β2 to tau is attributed to weak interaction with tau through its unique higher-order structure of CT constructed by the 13 amino acids insertion. Such information may provide a clue for understanding of the physiological significance of the two splice variants of GSK-3β and a new insight into the regulation of tau phosphorylation in central nervous system.
Pharmacology
Regular Articles
  • Yasutomo Hori, Kazuki Yoshioka, Kazutaka Kanai, Fumio Hoshi, Naoyuki I ...
    原稿種別: Regular Article
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 61-65
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Although transregulation between the sympathetic nervous system and the renin-angiotensin-aldosterone system has been reported, it remains unclear whether sympathetic hyperactivity-induced matrix metalloproteinease (MMP) expression/activity and cardiac fibrosis are mediated by the mineralocorticoid receptor system. We investigated whether isoproterenol (ISO)-induced MMP expression/activity and cardiac fibrosis are mediated by spironolactone in rats. Male Wistar Kyoto rats were divided into 3 groups: control, ISO, and ISO combined with spironolactone (SPI). ISO (2.0 mg/kg/d) and/or SPI (40 mg/kg/d) were given for 14 d. Echocardiography and hemodynamic measurements were recorded and hearts were excised. The myocyte cross-sectional and fibrotic area was evaluated via histopathological analysis. MMP-2 and collagen-I were analyzed by Western blotting and zymography. Compared with the controls, ISO significantly elevated the end-diastolic left ventricular (LV) pressure and the time constant of isovolumic relaxation and decreased the −dP/dt, while those of SPI co-treatment did not. ISO treatment induced significant increases in the fractional shortening and relative wall thickness, whereas SPI co-treatment significantly decreased relative wall thickness. Similarly, ISO significantly increased LV weight and myocyte cross-sectional and fibrotic area, which occurred concomitantly with the MMP-2 expression/activity and the expression of collagen-I. Moreover, ISO induced these features were significantly attenuated by SPI co-treatment. Our results suggest that ISO-evoked sympathetic hyperactivity induced LV fibrosis and MMP-2, which may be partially controlled via the mineralocorticoid receptor system.
  • Michael du Plooy, Michelle Viljoen, Malie Rheeders
    原稿種別: Regular Article
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 66-70
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    The interaction between verapamil, a P-glycoprotein (P-gp) inhibitor, with ritonavir and lopinavir/ritonavir (LPV/r) after acute and chronic treatment was investigated in rats. Rats were divided into 4 groups, viz. Group 1: ritonavir, 20 mg/kg/d (n=18), group 2: ritonavir, 20 mg/kg/d plus verapamil 5 mg/kg/d (n=18), group 3: LPV/r, 80 and 20 mg/kg/d (n=17) and group 4: LPV/r, 80 and 20 mg/kg/d plus verapamil 5 mg/kg/d (n=18). Blood samples were collected after decapitation on days 1, 7 and 21. Lopinavir and ritonavir plasma levels were simultaneous determined by a validated LC/MS/MS method. The lower limit of quantification for both ritonavir and lopinavir was 0.078 μg/ml. Verapamil significantly increased ritonavir plasma levels, administered as monotherapy, following acute (p<0.005) and chronic treatment (day 21) (p<0.005). During acute (but not chronic) LPV/r treatment, verapamil also increased the lopinavir levels (p<0.05). A time or exposure dependent pharmacokinetic interaction was thus observed between verapamil and ritonavir whether administered alone or after the lopinavir-ritonavir combination (LPV/r). This interaction occurred most prominently after acute treatment, and became less pronounced over time. This study indicates the importance of a longer time frame to investigate enzyme based drug interactions in rat models.
  • Mikihito Hayashi, Naoki Matsumoto, Sachiko Takenoshita-Nakaya, Yuko Ta ...
    原稿種別: Regular Article
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 71-76
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Some P450 enzymes are expressed not only in the liver but also in the small intestine, and these enzymes play an important role in first-pass drug metabolism in the small intestine. Cytochrome P450 (CYP)2C19 has been confirmed to exist in the small intestine of white people, but not yet in Japanese. We investigated the mRNA level, protein level, and activity of CYP2C19 in the small intestine in a Japanese population. Samples were obtained from the healthy portions of resected small intestines from 18 patients who had undergone pancreatoduodenectomy. The microsomes were extracted from the epithelium of the small intestinal tissues. CYP2C19 mRNA and protein levels were analyzed using real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CYP2C19 activity in the microsomes was evaluated based on the 5-hydroxylation of lansoprazole using HPLC. CYP2C19 mRNA and protein levels and activities in the small intestine showed interindividual differences. CYP2C19 mRNA levels were not correlated with protein levels or its activity. On the other hand, there was significant correlation between CYP2C19 protein levels and its activity. Further, CYP2C19 protein levels and activities in the small intestine were approximately equal to those in liver. These results suggest the metabolic capacity of CYP2C19 in Japanese small intestine may play as important a role as the liver in drug metabolism. Analyses of the protein level or protein activity of CYP2C19 rather than its mRNA level should be required for predicting the individual metabolic capacity of CYP2C19 in the small intestine.
  • Yoshihisa Kitamura, Maho Doi, Keiko Kuwatsuka, Yuka Onoue, Ikuko Miyaz ...
    原稿種別: Regular Article
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 77-81
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Adult hippocampal neurogenesis is reported to change in animal models of depression and antidepressants. We have used the mitotic marker 5-bromo-2′-deoxyyridine to address the effects of imipramine and lithium on cell proliferation and survival following chronic treatment with adrenocorticotropic hormone (ACTH) in the subgranular zone of the hippocampal dentate gyrus. ACTH treatment for 14 d decreased adult hippocampal cell proliferation and survival. Coadministration of imipramine and lithium for 14 d blocked the loss of cell proliferation but not cell survival resulting from the chronic treatment with ACTH. The coadministration of imipramine and lithium may have treatment-resistant antidepressive properties, which may be attributed, in part, to a normalization of hippocampal cell proliferation.
  • Eisaku Nakamura, Naoko Tanaka, Masachika Kuwabara, Atsushi Yamashita, ...
    原稿種別: Regular Article
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 82-86
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    It is established that the segment of saphenous vein (SV) that is widely used as a conduit vessel in coronary artery bypass graft (CABG) surgery is distended with high pressure to check for leaks and to increase the patency before implantation into coronary arterial circulation. The aim of the present study was to elucidate the relative contributions of 5-hydroxytryptamine (5-HT) receptor subtypes responsible for 5-HT-induced vasoconstriction of the distended human SV. Whereas about half of the 5-HT-induced vasoconstriction still remained in the presence of supramaximum concentration of sarpogrelate or of SB224289 (5-HT2A and 5-HT1B receptor antagonists, respectively), simultaneous treatment with sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. Immunopositive staining for 5-HT2A and 5-HT1B receptors was detected in smooth muscle cells of the distended human SV and there was no significant difference between the immunopositive areas of 5-HT2A and 5-HT1B receptors. These results demonstrate that 5-HT2A and 5-HT1B receptors similarly contribute to 5-HT-induced vasoconstriction in human distended SV. Thus, when the SV is used as a CABG conduit, a combination of 5-HT2A and 5-HT1B receptor antagonists would appear to be most useful to prevent 5-HT-induced spasm.
  • Mariko Fujita, Atsushi Momose, Takayuki Ohtomo, Azusa Nishinosono, Kou ...
    原稿種別: Regular Article
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 87-91
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    In rodent models of diet-induced obesity, prolonged high-fat feeding increases the cellular uptake of fatty acids and causes lipotoxicity in the heart and skeletal muscle, where substrate overload to beta-oxidation generates mitochondrial stress. We examined the hypothesis that, because of its catalytic properties, acyl-CoA thioesterase (ACOT) would counteract these detrimental situations by modulating intracellular acyl-CoA levels. Rats were fed a low- or high-fat diet for up to 20 weeks, and the expressions of ACOT isoforms and fatty acid beta-oxidation enzymes were analyzed by western blotting. The expressions of ACOT1, ACOT2 and ACOT7 proteins in the heart and soleus muscle were significantly increased, by 2.0—7.6-fold, in rats fed the high-fat diet as compared with the low-fat diet group. These effects were accompanied by increases in carnitine palmitoyltransferase and acyl-CoA oxidase expression. However, ACOT was not induced in the extensor digitorum longus muscle or the liver. Subcellular fractionation of heart and soleus muscle homogenates confirmed expression of both the cytosolic and mitochondrial ACOT isoforms. These results underscore the functional relationship between ACOT and fatty acid oxidation, and suggest adaptive upregulation of ACOT to protect against fatty acid oversupply in the heart and skeletal muscle.
  • Masatoshi Inden, Yoshihisa Kitamura, Mari Abe, Aya Tamaki, Kazuyuki Ta ...
    原稿種別: Regular Article
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 92-96
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Chronic systemic exposure of Lewis rats to rotenone produced many features of Parkinson's disease (PD), including nigrostriatal dopamine (DA) neurodegeneration and the formation of cytoplasmic inclusions in nigral DA neurons. We also reported that chronic oral administration of rotenone at 30 mg/kg for 28 d caused specific nigrostriatal DA neurodegeneration in C57BL/6 mice. To establish a PD model more suitable for evaluating nigrostriatal DA neurodegeneration, the present study has been designed to assess the neurotoxicity of rotenone after daily oral administration at 30 or 100 mg/kg for 56 d in C57BL/6 mice. The survival rate of rotenone-treated mice at 30 mg/kg did not change from 28 to 56 d, although the survival rate of rotenone-treated mice at 30 mg/kg was decreased to about 70% within one week. The survival rate of the rotenone-treated mice at 100 mg/kg was suddenly decreased after 28 d, and finally to about 15% at 56 d. Rotenone at 30 mg/kg, but not 100 mg/kg, for 28 d caused a significant loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. Rotenone at 100 mg/kg caused a highly variable loss of TH-positive neurons among individual mice. Rotenone at 30 mg/kg for 56 d caused a significant loss of TH-positive neurons and behavioral impairment. In addition, α-synuclein immunoreactivity was increased in surviving TH-positive neurons in a time-dependent manner. Thus, this protocol for chronic administration of rotenone at 30 mg/kg for 56 d is more useful for understanding the mechanism of DA neurodegeneration.
Notes
  • Asami Mori, Sachi Suzuki, Kenji Sakamoto, Tsutomu Nakahara, Kunio Ishi ...
    原稿種別: Note
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 150-152
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    The large-conductance Ca2+-activated K+ (BKCa) channels modulate vascular smooth muscle tone but the role of BKCa channels in regulation of retinal circulation remains unclear. In the present study, we examined the effects of BMS-191011 and NS 1619, openers of BKCa channels, on rat retinal blood vessels in vivo. Male Wistar rats (8- to 10-week-old) were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally (i.p.)) and treated with tetrodotoxin (50 μg/kg, intravenously (i.v.)) to eliminate any nerve activity and prevent movement of the eye under artificial ventilation. A mixture solution of adrenaline and noradrenaline (9 : 1) was infused to maintain adequate systemic circulation. BMS-191011 (10—100 μg/kg, i.v.) and NS 1619 (0.1—1.0 μg/kg, i.v.) increased the diameter of retinal arterioles without altering systemic blood pressure and heart rate significantly. The vasodilator responses to BMS-191011, but not to NS 1619, were significantly diminished by intravitreal injection of iberiotoxin (an inhibitor of BKCa channels, 20 pmol/eye). These results suggest that BMS-191011 dilates rat retinal arterioles through activation of iberiotoxin-sensitive BKCa channels in vivo. The BKCa channel opener could be considered as a candidate for improving retinal circulation without severe cardiovascular side-effects.
  • Iyuki Namekata, Noriko Tsuruoka, Yayoi Tsuneoka, Tomoyuki Matsuda, Aki ...
    原稿種別: Note
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 153-155
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    We examined the effect of NIP-142, a benzopyran compound with terminating effect on experimental atrial arrhythmia, on the KCNQ1/KCNE1 channel, which underlies the slow component of the cardiac delayed rectifier potassium channel (IKs). NIP-142, as well as chromanol 293B, showed concentration-dependent blockade of the current expressed in HEK293 cells; the EC50 value of NIP-142 and chromanol 293B for the inhibition of tail current was 13.2 μM and 4.9 μM, respectively. These results indicate that NIP-142 has blocking effect on the KCNQ1/KCNE1 channel current.
  • Kenji Sakamoto, Tsutomu Nakahara, Kunio Ishii
    原稿種別: Note
    専門分野: Pharmacology
    2011 年 34 巻 1 号 p. 156-159
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    It has been shown that p38 mitogen-activated protein (MAP) kinase is absolutely necessary for the cardioprotection of early ischemic preconditioning in the heart. Reorganization of actin cytoskeleton after translocation of HSP27, which is mediated by p38 MAP kinase, was reported to be necessary for the cardioprotective effect of early ischemic preconditioning. Although Rho and Rho kinase are reported to regulate reorganaization of actin filaments, it is unknown whether Rho–Rho kinase pathway is involved in the cardioprotective effect of early ischemic preconditioning. The aim of the present study is to determine the involvement of Rho–Rho kinase pathway in the protective effect of early ischemic preconditioning in the rat hearts. Dominant-negative Rho significantly reduced the hypoxia-reoxygenation-induced activation of p38 MAP kinase, and constitutive active Rho activated p38 MAP kinase in rat myoblast H9c2 cells. Y-27632, a specific Rho kinase inhibitor, concentration-dependently attenuated the post-ischemic recovery of left ventricular developed pressure by early ischemic preconditioning. Thus, Rho–Rho kinase pathway is, at least in part, involved in the mechanism of early ischemic preconditioning.
Pharmacognosy
Regular Articles
  • Gil-Saeng Jeong, Dong-Sung Lee, Mi-Young Song, Byung-Hyun Park, Dae-Gi ...
    原稿種別: Regular Article
    専門分野: Pharmacognosy
    2011 年 34 巻 1 号 p. 97-102
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Butein (3,4,2′,4′-tetrahydroxychalcone), a plant polyphenol, is a major component in isolate of Rhus verniciflua STOKES (Anacardiaceae). It is shown to exert various potent effects such as antioxidant, antiinflammatory induction of apoptosis among many properties. In this study, we investigated the effect of butein on cytokine-induced β-cell damage. Pre-treatment with butein is shown to increase the viability of cytokine-treated INS-1 cells at concentrations of 15—30 μM. Butein prevented cytokine-mediated cell death, as well as nitric oxide (NO) production, and these effects correlated well with reduced levels of protein expression of the inducible nitric oxide synthase (iNOS). Furthermore, the molecular mechanisms by which butein inhibits iNOS gene expression appeared to be through the inhibition of nuclear factor-κB (NF-κB) translocation. In a second set of experiments, rat islets were used to demonstrate the protective effects of butein and the results were essentially the same as those observed in Beutin pretreated INS-1 cells. Butein prevented cytokine-induced NO production, iNOS expression, and NF-κB translocation and inhibition of glucose-stimulated insulin secretion (GSIS). In conclusion, these results suggest that butein can be used for the prevention of functional β-cell damage and preventing the progression of Type 1 diabetes mellitus (T1DM).
  • Kanako Okusada, Kazuo Nakamoto, Mikako Nishida, Wakako Fujita-Hamabe, ...
    原稿種別: Regular Article
    専門分野: Pharmacognosy
    2011 年 34 巻 1 号 p. 103-107
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Morinda citrifolia (Rubiaceae, Noni) is a traditional medicine with various pharmacological activities. We investigated if the MeOH-, CHCl3- and BuOH-soluble phase and its main active component, damnacanthal, isolated from the Noni root, have antinociceptive and anti-inflammatory actions in mice. The CHCl3-soluble phase (3 g/kg, per os (p.o.)) significantly reduced pain-related behavior observed in the formalin test. These effects were not suppressed by pretreatment with naloxone (1 mg/kg, intraperitoneally (i.p.)), an opioid receptor antagonist. The CHCl3-soluble phase (3 g/kg, p.o.) significantly reduced histamine-induced paw edema. The MeOH- and BuOH-soluble phase had no effect in either test. Furthermore, damnacanthal (10—100 mg/kg, p.o.) exerted an antinociceptive effect on chemical nociceptive stimuli, and decreased histamine-induced paw edema. Damnacanthal was weakly bound to the histamine H1 receptor. These data suggest that the CHCl3-soluble phase of the Noni root has antinociceptive and anti-inflammatory effects. Furthermore, these effects of damnacanthal isolated from the Noni root is mediated in part by the histamine H1 receptor.
Note
  • Jin Kyu Kim, Yeon Sil Lee, Seon Ha Kim, Young Soo Bae, Soon Sung Lim
    原稿種別: Note
    専門分野: Pharmacognosy
    2011 年 34 巻 1 号 p. 160-163
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Aldose reductase (AR) inhibitors have considerable therapeutic potential against diabetic complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of the 70% acetone extract obtained from Paulownia coreana seeds, phenylpropanoid glycosides (compounds 1—4) and 5 phenolic coumpounds were isolated (compounds 5—9). Their structures were determined on the basis of spectroscopic analysis and comparison with reported data. All the isolates were subjected to in vitro bioassays to evaluate their inhibitory activities against recombinant human aldose reductase (rhAR) and sorbitol formation in human erythrocytes. Phenylethanoid glycosides showed more effective than the phenolic compounds in inhibiting rhAR. Among the compounds, isocampneoside II (3) was found to significantly inhibit rhAR with an IC50 value of 9.72 μM. In kinetic analyses performed using Lineweaver–Burk plots of 1/velocity and 1/concentration of substrate, isocampneoside II (3) showed uncompetitive inhibition against rhAR. Furthermore, it inhibited sorbitol formation in a rat lens incubated with a high concentration of glucose; this finding indicated that isocampneoside II (3) may effectively prevent osmotic stress in hyperglycemia. Thus, the P. coreana-derived phenylethanoid glycoside isocampneoside II (3) may have a potential therapeutics against diabetic complications.
Biopharmacy
Regular Articles
  • Satoshi Ueshima, Tetsuya Aiba, Tomoaki Sato, Hisashi Matsunaga, Yuji K ...
    原稿種別: Regular Article
    専門分野: Biopharmacy
    2011 年 34 巻 1 号 p. 108-113
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    The unbound serum concentration of valproic acid (VPA) is closely related to its therapeutic efficacy. In epileptic infants, the unbound VPA concentration varies largely from patient to patient, being difficult to predict using the reported equations for older children. To establish an equation to estimate the unbound concentration in infants, we empirically characterized the relationship between total and unbound VPA concentrations, taking their growth and development into consideration. Data were retrospectively collected from archived clinical records of 30 epileptic infants aged 0—11 months old. The relationship between total and unbound VPA concentrations was analyzed according to the Langmuir equation, in which the patient's body weight, height, and body surface area were considered as physical development indices. Inter- and intra-individual variabilities in the VPA concentrations were also considered. It was shown that the unbound VPA concentration in infants is properly estimated when their body weights are taken into account, in which the parameter for the maximum binding site concentration (Bm) increases as the body weight increases, while that for the dissociation constant (Kd) is unaltered. Additionally, the relationship was shown to slightly change when the infants are concomitantly treated with VPA and the other antiepileptics. These findings provide useful information to adjust the VPA dosage to achieve optimal therapeutic efficacy in epileptic infants.
  • Takeru Nambu, Akinobu Hamada, Reiko Nakashima, Misato Yuki, Tatsuya Ka ...
    原稿種別: Regular Article
    専門分野: Biopharmacy
    2011 年 34 巻 1 号 p. 114-119
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Intracellular concentration of imatinib in leukemic cells is thought to affect the clinical efficacy of this drug in patients with chronic myeloid leukemia (CML); however, there is no report that directly indicates the relationship between intracellular concentration and clinical outcome and/or, plasma concentration. In addition, the impacts of genetic variations of drug transporters, which mediate leukocyte concentration of imatinib, are unknown. In the present study, we investigated the correlation between intracellular imatinib concentrations in leukocytes, plasma imatinib levels, and genotypes of drug transporters, including ATP binding cassette B1 (ABCB), ABCG2, solute carrier 22A1 (SLC22A1), solute carrier organic anion transporter family members 1B1 (SLCO1B1) and SLCO1B3. The imatinib levels in leukocytes were determined using HPLC in 15 patients with chronic phase CML. No significant correlation between intracellular and plasma concentrations of imatinib was observed. The intracellular concentration was comparable in both patients with or without complete cytogenetic response. The intracellular imatinib concentration was significantly higher in patients with SLCO1B3 334TT than in those with 334TG/GG (p=0.0188). Plasma concentrations were similar in both SLCO1B3 genotypes (p=0.860), thereby resulting in the intracellular to plasma concentration ratio being higher in patients with SLCO1B3 334TT than those with 334 TG/GG (p=0.0502). These results suggested that the SLCO1B3 334T>G polymorphism could have a significant impact on the intracellular concentration of imatinib in leukocytes as a promising biomarker for personalized treatment of CML patients.
Note
  • Shozo Miyazaki, Mariko Ishitani, Akie Takahashi, Tetsuya Shimoyama, Ku ...
    原稿種別: Note
    専門分野: Biopharmacy
    2011 年 34 巻 1 号 p. 164-166
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    We have designed gel formulations for the oral administration of acetaminophen with suitable rheological characteristics for ease of administration to patients with swallowing difficulties and sufficient integrity in the stomach to achieve a sustained release of this drug. Gels formed by agar and ι-carrageenan were assessed for suitable gel strength and in vitro and in vivo release characteristics. Comparison of 1.5% ι-carrageenan gel with 0.5% agar gel demonstrated improved sustained release properties of the ι-carrageenan gel. Gel formed by ι-carrageenan has suitable rheological and sustained release characteristics for potential use as vehicles for oral delivery of drugs to dysphagic patients.
Miscellaneous
Regular Articles
  • Hye Young Kim, Jin Hee Choi, Young Jin Kang, So Young Park, Hyoung Chu ...
    原稿種別: Regular Article
    専門分野: Miscellaneous
    2011 年 34 巻 1 号 p. 120-127
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Reparixin, an inhibitor of CXCL8 receptor CXCR1 and CXCR2 activation, has been shown to attenuate inflammatory responses in various injury models. In the present study, the hypertension-related functional roles of reparixin were examined in hypertensive animals. Spontaneously hypertensive rats (SHR) at the age of 18 weeks were administered a subcutaneous injection of reparixin (5 mg/kg) daily for 3 weeks (SHR-R, n=5). Control groups consisted of normal saline-treated SHR (SHR-N, n=5) and normotensive Wistar–Kyoto rats (WKY-N, n=5). Reparixin effectively decreased systolic blood pressure and increased the blood flow. The thoracic aorta wall thickness was significantly decreased in SHR-R compared to SHR-N. Expressions of CXCL8, CCL2, 12-lipoxygenase (LO) and endothelin (ET)-1 were significantly decreased in SHR-R thoracic aorta tissues compared to SHR-N. Furthermore, expression of angiotensin II subtype I receptor (AT1R) protein was decreased in SHR-R thoracic aorta tissues compared to SHR-N. In addition, the plasma levels of nitric oxide were slightly elevated in SHR-R compared to the levels in SHR-N. These findings indicate that inhibition of hypertension-related mediators by reparixin results in the reduction of blood pressure in SHR. Therefore, these results suggest that reparixin-mediated blockade of CXCL8 receptor activation attenuates vascular hypertension in SHR.
  • Haruyo Iwasawa, Erika Morita, Satoru Yui, Masatoshi Yamazaki
    原稿種別: Regular Article
    専門分野: Miscellaneous
    2011 年 34 巻 1 号 p. 128-134
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    We previously reported that kiwi fruit is rich in polyphenols and has immunostimulatory activity. Polyphenols are widely known for having anti-oxidant effects. We also revealed potential anti-oxidant effects of kiwi fruit in vivo by oral administration to mice. Here, we compared the anti-oxidant effects of kiwi fruit with those of other fruits in vitro. Then, we examined the inhibitory effects of kiwi fruit on oxidation in the human body. There are two varieties of kiwi fruit, green kiwi and gold kiwi. We also examined variation between these varieties. Comparison of the anti-oxidant effects in vitro demonstrated that kiwi fruit had stronger anti-oxidant effects than orange and grapefruit, which are rich in vitamin C; gold kiwi had the strongest anti-oxidant effects. Kiwi fruit inhibited oxidation of biological substances in the human body. In particular, kiwi fruit may inhibit early lipid oxidation. In this study, kiwi fruit had strong anti-oxidant effects and may prevent the development and deterioration of diseases caused by oxidative stress.
Notes
  • Maria Jose Herrero, Daniel Monleon, Jose Manuel Morales, Manuel Mata, ...
    原稿種別: Note
    専門分野: Miscellaneous
    2011 年 34 巻 1 号 p. 167-172
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    The hydrodynamic injection in mice tail vein of a plasmid (40 μg DNA) bearing the human α1-antitrypsin gene mediates: a) good liver gene transfer resulting in therapeutic plasma levels of human protein (1 mg/ml, approximately) from days 1—10 after injection; b) low liver injury as demonstrated by a poor and transient increase of aspartate aminotransferase (AST) and alanine transaminase (ALT) in mouse plasma; 3) limited expression and metabolic changes in host liver genes and metabolites as evaluated on days 2 and 10 after injection. Groups of three mice were uninjected (control) or hydrodynamically injected with saline or plasmid DNA and then sacrificed on days 2 and 10 after injection. The results of principal component analysis (PCA) show, both in expression microarray and metabolomic anaylisis, that changes between control and hydrodinamically injected groups are not dramatic and tend to normalize after 10 d. The differences are even smaller between DNA and saline hydrodinamically injected mice. Hydrodynamic injection induces a complex but limited gene expression and metabolic change which includes variations in molecules related to energy metabolism and stress response. The results contribute to support that hydrodynamic method is a safe procedure of liver gene transfer but the long-term effect of hydrodynamic gene transfer procedure, remains to be studied.
  • Mahmoud Kandeel, Yukio Kitade
    原稿種別: Note
    専門分野: Miscellaneous
    2011 年 34 巻 1 号 p. 173-176
    発行日: 2011/01/01
    公開日: 2011/01/01
    ジャーナル フリー
    Plasmodium falciparum thymidylate kinase (PfTMK) is a potential chemotherapeutic target as it can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. An important step in drug development is to determine the interaction of ligands competing for their target sites in a proposed drug target. The estimated binding affinity of thymidylate (TMP) with PfTMK/deoxyguanylate complex was as low as 0.9×104 M−1 with a very low exothermic signal of −3.9 kcal mol−1. Furthermore, titration of PfTMK/TMP with deoxyguanylate (dGMP) showed a very small heat signal corresponding to nonspecific background heat. Titration of PfTMK with a 1 : 1 mixture of TMP and dGMP showed a binding affinity corresponding to the average of the binding affinity for individual reactions. Thus, dGMP was unable to displace TMP from its binding site, while TMP was able to partially displace dGMP from the its binding site accompanied by a weak exothermic signal and lowered affinity. Based on these results, we propose that pyrimidine based inhibitors will compete with TMP and may be able to more efficiently displace dGMP from binding sites compared with purine based compounds. In addition, the synthesis of purine based compounds as inhibitors of PfTMK will be highly selective for the parasitic enzyme, however, they need to be potent enough to displace TMP from its binding site.
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