56 results on '"Homma M"'
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2. Structure of MotA, a flagellar stator protein, from hyperthermophile.
- Author
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Nishikino T, Takekawa N, Tran DP, Kishikawa JI, Hirose M, Onoe S, Kojima S, Homma M, Kitao A, Kato T, and Imada K
- Subjects
- Archaea metabolism, Bacteria metabolism, Membrane Proteins metabolism, Molecular Motor Proteins metabolism, Bacterial Proteins metabolism, Flagella chemistry
- Abstract
Many motile bacteria swim and swarm toward favorable environments using the flagellum, which is rotated by a motor embedded in the inner membrane. The motor is composed of the rotor and the stator, and the motor torque is generated by the change of the interaction between the rotor and the stator induced by the ion flow through the stator. A stator unit consists of two types of membrane proteins termed A and B. Recent cryo-EM studies on the stators from mesophiles revealed that the stator consists of five A and two B subunits, whereas the low-resolution EM analysis showed that purified hyperthermophilic MotA forms a tetramer. To clarify the assembly formation and factors enhancing thermostability of the hyperthermophilic stator, we determined the cryo-EM structure of MotA from Aquifex aeolicus (Aa-MotA), a hyperthermophilic bacterium, at 3.42 Å resolution. Aa-MotA forms a pentamer with pseudo C5 symmetry. A simulated model of the Aa-MotA
5 MotB2 stator complex resembles the structures of mesophilic stator complexes, suggesting that Aa-MotA can assemble into a pentamer equivalent to the stator complex without MotB. The distribution of hydrophobic residues of MotA pentamers suggests that the extremely hydrophobic nature in the subunit boundary and the transmembrane region is a key factor to stabilize hyperthermophilic Aa-MotA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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3. Reduction of tumor hypoxia by anti-PD-1 therapy assessed using pimonidazole and [ 18 F]FMISO.
- Author
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Nakajima K, Homma M, Suzuki M, Yokouchi Y, Matsuda T, Takakura H, Hirata K, Kuge Y, and Ogawa M
- Subjects
- Animals, CD8-Positive T-Lymphocytes, Cell Hypoxia, Hypoxia, Mice, Misonidazole analogs & derivatives, Nitroimidazoles, Positron-Emission Tomography methods, Tumor Microenvironment, Neoplasms, Tumor Hypoxia
- Abstract
Introduction: Hypoxia is common in solid tumors and creates an immunosuppressive environment that leads to resistance to immunotherapy, such as an anti-programmed death receptor-1 (PD-1) therapy. It has been suggested that anti-PD-1 therapy may reduce tumor hypoxia by remodeling the tumor vasculature; however, it is unclear how anti-PD-1 therapy reduces hypoxia over time. Therefore, we investigated the relationship between hypoxia and immune activation by anti-PD-1 therapy in murine cancer models., Methods: Anti-PD-1 antibody was injected to CT26- and MC38-tumor-bearing mice on days 0 and 5. Tumor hypoxia was non-invasively evaluated using positron emission tomography (PET) with [
18 F]fluoromisonidazole ([18 F]FMISO) on days 3 and 7. Histological analysis was conducted to investigate the infiltration of immune cells in [18 F]FMISO-accumulated hypoxic area. In addition, the immune cell population in tumors and the percentages of cancer and immune cells under hypoxic conditions were analyzed at single-cell level using flow cytometry., Results: Flow cytometric analysis of CT26 tumors on day 3 showed that anti-PD-1 therapy reduced hypoxia without inhibition of tumor growth. In addition, the infiltration of CD8+ T cells was increased in treated tumors. In contrast to CT26 tumors, the percentage of hypoxic cells in MC38 tumors did not change on days 3 and 7, and there was minimal immune activation induced by anti-PD-1 antibody. Changes in hypoxia in CT26 tumors were not detected by [18 F]FMISO-PET, but autoradiogram showed that [18 F]FMISO accumulated in immunosuppressed areas, where the infiltration of immune cells was relatively low., Conclusion: Reduction of hypoxia was induced in CT26 tumor, in which adequate immune response to anti-PD-1 therapy was exhibited, at an early time point before suppression of tumor growth. Our findings suggest that anti-PD-1 therapy can create a tumor microenvironment that facilitates immune activation by reducing hypoxia., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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4. The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos.
- Author
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Fan W, Homma M, Xu R, Kunii H, Bai H, Kawahara M, Kawaguchi T, and Takahashi M
- Subjects
- Animals, Apoptosis genetics, Blastocyst cytology, Blastomeres cytology, DNA Fragmentation, Endopeptidase K metabolism, Female, In Situ Nick-End Labeling methods, Isotonic Solutions chemistry, Male, Mice, Inbred ICR, Microscopy, Fluorescence methods, Mice, Blastocyst physiology, Blastomeres physiology, Embryo Culture Techniques methods, Embryonic Development physiology, Zona Pellucida physiology
- Abstract
The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos. We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system., Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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5. Effects of an Indomethacin Oral Spray on Pain Due to Oral Mucositis in Cancer Patients Treated With Radiotherapy and Chemotherapy: A Double-Blind, Randomized, Placebo-Controlled Trial (JORTC-PAL04).
- Author
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Nagaoka H, Momo K, Hamano J, Miyaji T, Oyamada S, Kawaguchi T, Homma M, Yamaguchi T, Morita T, and Kizawa Y
- Subjects
- Adult, Double-Blind Method, Humans, Indomethacin, Oral Sprays, Pain drug therapy, Pain etiology, Quality of Life, Head and Neck Neoplasms, Stomatitis drug therapy, Stomatitis etiology
- Abstract
Context: Oral mucositis (OM) pain due to anticancer chemo- and radiotherapy has a very negative impact on patient quality of life. However, no high-quality studies have been performed regarding the analgesic efficacy of indomethacin (IM) oral spray for OM pain., Objectives: This randomized, placebo-controlled, double-blind trial aimed to evaluate the analgesic efficacy of IM oral spray for OM pain due to anticancer chemo- and radiotherapy., Methods: From July 2015 to December 2016, we enrolled adult cancer patients with OM pain that was due to anticancer chemo- or radiotherapy and was rated 4 or higher on Brief Pain Inventory (BPI) Item 5. Patients were randomly assigned in a 1:1 ratio to receive either IM oral spray or placebo. The primary endpoint was the change in the BPI Item 6 ("current pain") score from before to 30 minutes after treatment. Secondary endpoints were the areas under the curves of BPI Item 6 at 15, 60, 120, 180, and 240 minutes after treatment; five items related to meals and conversation from the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire, Head and Neck Module 35; the Clinical Global Impressions-Improvement (CGI-I) scale; and adverse events., Results: A total of 60 patients were assigned to receive IM oral spray (n = 33) or placebo spray (n = 27). The average change in the BPI item 6 score from before to 30 minutes after treatment was -1.85 (95% confidence interval: -2.37 to -1.32) in the IM spray group and -0.59 (-1.02 to -0.16) in the placebo group, indicating a significant difference (-1.26, -1.94 to -0.57, P < 0.01). The pain improvement persisted for 180 minutes. The intergroup differences in ability to drink liquids, ease in conversing, and CGI-I were all significant (P = 0.03, P = 0.02, and P < 0.01, respectively). No serious adverse events were reported., Conclusion: IM oral spray alleviated short-term OM pain due to anticancer chemo- and radiotherapy, and may reduce the difficulty in eating meals., (Copyright © 2021 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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6. Phase 1 study of pembrolizumab plus chemotherapy as first-line treatment in Japanese patients with advanced NSCLC.
- Author
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Kurata T, Nakagawa K, Satouchi M, Seto T, Sawada T, Han S, Homma M, Noguchi K, and Nogami N
- Subjects
- Adult, Aged, Antibodies, Monoclonal, Humanized pharmacology, Antineoplastic Combined Chemotherapy Protocols pharmacology, Carcinoma, Non-Small-Cell Lung pathology, Female, Humans, Japan, Lung Neoplasms pathology, Male, Middle Aged, Antibodies, Monoclonal, Humanized therapeutic use, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Carcinoma, Non-Small-Cell Lung drug therapy, Lung Neoplasms drug therapy
- Abstract
Introduction: Pembrolizumab plus chemotherapy significantly improved outcomes over chemotherapy alone as first-line treatment in patients with advanced non-small-cell lung cancer (NSCLC) in phase 3 international trials. In the phase 1 KEYNOTE-011 study (parts B and C), we evaluated the safety/activity of pembrolizumab plus chemotherapy as first-line treatment in Japanese patients with advanced NSCLC., Methods: Eligible patients received 4 cycles (every 3 weeks) of pembrolizumab 200 mg plus chemotherapy (cisplatin 75 mg/m
2 /carboplatin area under the curve [AUC] 5 mg/mL/min and pemetrexed 500 mg/m2 in part B [nonsquamous]; carboplatin AUC 6 mg/mL/min and paclitaxel 200 mg/m2 /nab-paclitaxel 100 mg/m2 (weekly) in part C [squamous]), followed by maintenance pembrolizumab (and pemetrexed, part B). The primary endpoint was incidence of dose-limiting toxicities (DLTs) during the first 3 weeks of treatment. Overall response rate (ORR; RECIST v1.1 by central review) was exploratory., Results: In part B (median follow-up, 16.0 months; n = 12) 1 DLT occurred (grade 4 hyponatremia). Grade ≥3 treatment-related adverse events (AEs) occurred in 9 patients (75%). Two patients had grade 5 treatment-related AEs (pneumonitis and interstitial lung disease). In part C (median follow-up, 9.9 months; n = 14), 2 DLTs occurred (both grade 3 febrile neutropenia). Grade ≥3 treatment-related AEs occurred in 11 patients (79%); none were fatal. ORR was 73% in part B and 50% in part C, irrespective of PD-L1 status., Conclusion: Safety results show first-line pembrolizumab plus chemotherapy is feasible in Japanese patients with advanced NSCLC. Antitumor activity was observed irrespective of PD-L1 status and was comparable to that in international studies., Trial Register: ClinicalTrials.gov, NCT01840579., (Copyright © 2021 Merck Sharp & Dohme Corp., The Author(s). Published by Elsevier Ltd.. All rights reserved.)- Published
- 2021
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7. Illness perceptions and negative responses from medical professionals in patients with fibromyalgia: Association with patient satisfaction and number of hospital visits.
- Author
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Homma M, Ishikawa H, and Kiuchi T
- Subjects
- Adult, Communication, Female, Humans, Japan, Male, Middle Aged, Office Visits, Perception, Surveys and Questionnaires, Attitude of Health Personnel, Fibromyalgia psychology, Patient Satisfaction, Physician-Patient Relations, Physicians psychology
- Abstract
Objective: To examine whether illness perceptions among patients with fibromyalgia and negative responses from medical professionals correlate with their satisfaction with their physicians or with their number of hospital visits., Methods: Questionnaires were sent by post to members of the Japan Fibromyalgia Support Association. Measures collected included, as independent variables, the Brief Illness Perception Questionnaire and the Illness Invalidation Inventory; and as outcomes, the Patient Satisfaction Consultation Questionnaire and the number of hospital visits., Results: We analyzed data from 304 patients. Multiple logistic regressions showed that perception of poor treatment control and the experience of being discounted and misunderstood by medical professionals were strongly correlated with dissatisfaction with attending physicians. Patients who perceived poor treatment control visited the hospital fewer times, while patients who reported being discounted by medical professionals visited more times. Patients' negative emotions correlated neither with patient satisfaction nor with the number of hospital visits., Conclusion: Treatment effectiveness and the respect accorded to patients were the key factors significantly correlated both with patient satisfaction and the number of hospital visits., Practice Implications: Physicians should not emphasize only patients' negative psychological status but also should convey a respectful attitude and help patients understand their current treatment is useful., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2018
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8. Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering.
- Author
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Liew CW, Hynson RM, Ganuelas LA, Shah-Mohammadi N, Duff AP, Kojima S, Homma M, and Lee LK
- Subjects
- Amino Acid Sequence, Bacterial Outer Membrane Proteins genetics, Bacterial Proteins genetics, Models, Molecular, Molecular Motor Proteins genetics, Protein Domains, Protein Structure, Quaternary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Salmonella enterica chemistry, Salmonella enterica genetics, Scattering, Small Angle, Sequence Homology, Amino Acid, Solutions, Vibrio alginolyticus chemistry, Vibrio alginolyticus genetics, X-Ray Diffraction, Bacterial Outer Membrane Proteins chemistry, Bacterial Proteins chemistry, Flagella chemistry, Molecular Motor Proteins chemistry
- Abstract
The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotB
C2 ) and Vibrio alginolyticus (PomBC5 ) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotBC2 , the dimeric conformation of the PomBC5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2018
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9. Systematic review and two new cases of primary upper urinary tract neuroendocrine carcinomas.
- Author
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Nakasato T, Hiramatsu A, Matsui Y, Unoki T, Shimoyama H, Oshinomi K, Morita J, Maeda Y, Naoe M, Fuji K, Ogura H, Homma M, Yamochi T, Takimoto M, and Ogawa Y
- Subjects
- Aged, Aged, 80 and over, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Carcinoma, Large Cell mortality, Carcinoma, Neuroendocrine mortality, Carcinoma, Small Cell mortality, Fatal Outcome, Female, Humans, Kidney Neoplasms mortality, Male, Middle Aged, Neoplasm Recurrence, Local, Ureteral Neoplasms mortality, Carcinoma, Large Cell drug therapy, Carcinoma, Neuroendocrine drug therapy, Carcinoma, Small Cell drug therapy, Kidney Neoplasms drug therapy, Ureteral Neoplasms drug therapy
- Abstract
Background: Upper urinary tract neuroendocrine carcinoma (UUT-NEC) is extremely rare and has a poor prognosis. Although a few cases of successful treatment have been reported, no treatment has shown established efficacy., Patients and Methods: We analyzed 70 UUT-NEC patients, including 68 with small cell neuroendocrine carcinoma (SCNEC) and large cell neuroendocrine carcinoma (LCNEC) reported between 1985 and 2017 and 2 treated at our hospital., Results: Median patient age was 66 years, 58.6% were men, and 60% were of Asian descent. Most UUT-NECs were SCNEC (68; 95.7%), whereas LCNEC was very rare (2; 2.9%). More than half of the patients had accompanying other histological components, the most common being urothelial carcinoma (51.5%), whereas 41.4% had NEC alone. Of the 70 patients, 27 underwent additional therapy (e.g., chemotherapy and radiotherapy) along with surgery. Median survival was 15 months. In univariate analysis, stages T1-2 disease showed better prognosis than stages T3-4 (P < 0.001). Additional treatment (e.g., chemotherapy and radiotherapy) significantly improved prognosis (P = 0.014). Moreover, platinum-based chemotherapy also was associated with improved prognosis (P = 0.017). For platinum-based chemotherapy, multicollinearity with additional treatments was strong, and, thus, these data were not included in the analysis. Multivariate analysis revealed pathological stage (T1-2 vs. T3-4; P = 0.003) and additional treatment (P = 0.028) to be independent predictors of improved prognosis., Conclusion: Although UUT-NEC has a poor prognosis, additional treatment along with surgery and therapeutic intervention and stage T1-2 disease are independent factors to improve prognosis., (Copyright © 2018. Published by Elsevier Ltd.)
- Published
- 2018
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10. Effect of dose timing on the blood concentration of lapatinib in patients with breast cancer.
- Author
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Ohgami M, Bando H, Ishiguro H, Tsuda M, Toriguchi N, Aogi K, Toi M, Masuda N, Mitsuhashi S, Kurosawa A, and Homma M
- Subjects
- Administration, Metronomic, Adult, Aged, Drug Administration Schedule, Female, Humans, Lapatinib, Middle Aged, Protein Kinase Inhibitors administration & dosage, Protein Kinase Inhibitors blood, Breast Neoplasms blood, Breast Neoplasms drug therapy, Quinazolines administration & dosage, Quinazolines blood
- Published
- 2017
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11. Serum flecainide S/R ratio reflects the CYP2D6 genotype and changes in CYP2D6 activity.
- Author
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Doki K, Sekiguchi Y, Kuga K, Aonuma K, and Homma M
- Subjects
- Anti-Arrhythmia Agents therapeutic use, Bepridil, Bignoniaceae genetics, DNA, Complementary genetics, Female, Genotype, Humans, Male, Microsomes, Liver drug effects, Microsomes, Liver enzymology, Middle Aged, Pharmacogenetics methods, Tachycardia blood, Tachycardia drug therapy, Tachycardia genetics, Tachycardia, Supraventricular blood, Tachycardia, Supraventricular drug therapy, Tachycardia, Supraventricular genetics, Cytochrome P-450 CYP2D6 genetics, Flecainide blood
- Abstract
The aims of this study were to clarify whether the ratio of S- to R-flecainide (S/R ratio) in the serum flecainide concentration was associated with the stereoselectivity of flecainide metabolism, and to investigate the effects of the cytochrome P450 (CYP) 2D6 (CYP2D6) genotype and CYP2D6 inhibitor on the serum flecainide S/R ratio. In vitro studies using human liver microsomes and cDNA-expressed CYP isoforms suggested that variability in the serum flecainide S/R ratio was associated with the stereoselectivity of CYP2D6-mediated flecainide metabolism. We examined the serum flecainide S/R ratio in 143 patients with supraventricular tachyarrhythmia. The S/R ratio was significantly lower in intermediate metabolizers and poor metabolizers (IMs/PMs) than in extensive metabolizers (EMs) identified by the CYP2D6 genotype. The cut-off value for the S/R ratio to allow the discrimination between CYP2D6 EMs and IMs/PMs was 0.99. The S/R ratio in patients with co-administration of bepridil, a potent CYP2D6 inhibitor, was lower than 0.99, regardless of the CYP2D6 genotype status. Other factors, including age, sex, body weight, and renal function, did not affect the serum flecainide S/R ratio. This study suggests that the serum flecainide S/R ratio reflects the CYP2D6 genotype and changes in CYP2D6 activity on co-administration of a CYP2D6 inhibitor., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2015
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12. Aprepitant does not alter prednisolone pharmacokinetics in patients treated with R-CHOP.
- Author
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Maie K, Okoshi Y, Takaiwa N, Kurita N, Hasegawa Y, Homma M, Ishii K, Kohda Y, and Chiba S
- Subjects
- Antibodies, Monoclonal, Murine-Derived administration & dosage, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Aprepitant, Cyclophosphamide administration & dosage, Doxorubicin administration & dosage, Drug Interactions, Humans, Lymphoma, Non-Hodgkin drug therapy, Morpholines pharmacology, Nausea chemically induced, Prednisolone administration & dosage, Rituximab, Vincristine administration & dosage, Antiemetics therapeutic use, Antineoplastic Combined Chemotherapy Protocols adverse effects, Morpholines therapeutic use, Nausea drug therapy, Prednisolone pharmacokinetics
- Published
- 2014
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13. Effects of dipyridamole coadministration on the pharmacokinetics of ribavirin in healthy volunteers.
- Author
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Suzuki Y, Homma M, Abei M, Hyodo I, and Kohda Y
- Subjects
- Adult, Cross-Over Studies, Dipyridamole administration & dosage, Equilibrative Nucleoside Transporter 1 antagonists & inhibitors, Equilibrative Nucleoside Transporter 1 biosynthesis, Healthy Volunteers, Humans, Male, RNA, Messenger metabolism, Ribavirin administration & dosage, Ribavirin blood, Dipyridamole pharmacology, Ribavirin pharmacokinetics
- Abstract
Ribavirin (RBV), a guanosine analog for treatment of hepatitis C, is a substrate of a nucleoside transporter, solute carrier family 29 member 1 (SLC29A1). To clarify the impact of SLC29A1 on the pharmacokinetics of RBV, an open-label, crossover study of single-dose RBV (200 mg, p.o.) with and without coadministration of dipyridamole (DP), an inhibitor of SLC29A1, was performed. Plasma and erythrocyte concentrations of RBV in the control phase and DP phase (25 mg, 3 times daily for 4 days) were compared in 10 healthy volunteers. SLC29A1 mRNA expression in peripheral blood mononuclear cells was also determined. In the DP phase, area under the concentration-time curves (AUC) of RBV in plasma and erythrocytes showed reductions of 23% and 17%, respectively (p < 0.05), with increases in apparent oral clearance of 18% and 25%, respectively (p < 0.05). The reduction rate of the AUC of erythrocyte RBV in the DP phase was associated with SLC29A1 mRNA expression: higher mRNA expression showed greater AUC reduction. The elimination half-life of both plasma and erythrocyte RBV did not differ between the 2 phases. These results suggest that RBV/DP coadministration reduces the concentration of RBV in blood by inhibiting an important role of SLC29A1 in gastrointestinal absorption of RBV.
- Published
- 2013
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14. Effect of anti-inflammatory supplementation with whey peptide and exercise therapy in patients with COPD.
- Author
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Sugawara K, Takahashi H, Kashiwagura T, Yamada K, Yanagida S, Homma M, Dairiki K, Sasaki H, Kawagoshi A, Satake M, and Shioya T
- Subjects
- Aged, Combined Modality Therapy methods, Energy Intake physiology, Female, Humans, Male, Quality of Life, Treatment Outcome, Weight Gain physiology, Whey Proteins, Anti-Inflammatory Agents administration & dosage, Dietary Supplements, Exercise Therapy methods, Milk Proteins administration & dosage, Pulmonary Disease, Chronic Obstructive therapy
- Abstract
Background: One of the major pathophysiologies in advanced chronic obstructive pulmonary disease (COPD) has been attributed to systemic inflammation. Meta-analysis of the 2005 Cochrane Database concluded the effect of nutritional supplementation alone on stable COPD was insufficient to promote body weight gain or exercise capacity. The aim of this study was to investigate the effectiveness of nutritional supplementation therapy using a nutritional supplement containing whey peptide with low-intensity exercise therapy in stable elderly patients with COPD., Method: In stable elderly COPD patients with %IBW and %FEV(1) of less than 110 and 80%, respectively, anti-inflammatory nutritional supplementation therapy was added to low-intensity exercise therapy. Thirty-six COPD patients were divided into those with and those without the ingestion of an anti-inflammatory nutritional supplement containing whey peptide, which exhibited an anti-inflammatory effect. These two groups were designated as the nutritional support and the control groups, respectively. The body composition, skeletal muscle strength, exercise tolerance, health-related QOL (HRQOL), and inflammatory cytokines were evaluated before and three months after nutritional support combined with exercise therapy in both the nutritional support group and the control group., Results: In the nutritional support group, the body weight, %IBW, FM, energy intake, %AC, Alb, PImax, PEmax, 6MWD, WBI, emotional function, and CRQ total were significantly increased, and the levels of hsCRP, IL-6, IL-8, and TNF-α were reduced significantly, while no significant change was noted in any item of physiological evaluation or any biomarker in the control group., Conclusion: Concomitant use of a anti-inflammatory nutritional supplement containing whey peptide, which exhibits an anti-inflammatory effect, with exercise therapy in stable elderly COPD patients with %IBW<110% and %FEV(1)<80% may not only increase body weight but may also inhibit systemic inflammation and thus improve exercise tolerance and HRQOL., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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15. Mutations targeting the C-terminal domain of FliG can disrupt motor assembly in the Na(+)-driven flagella of Vibrio alginolyticus.
- Author
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Kojima S, Nonoyama N, Takekawa N, Fukuoka H, and Homma M
- Subjects
- Mutagenesis, Site-Directed, Vibrio Infections metabolism, Vibrio Infections microbiology, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cell Movement physiology, Flagella physiology, Mutation genetics, Sodium metabolism, Sodium Channels metabolism, Vibrio alginolyticus physiology
- Abstract
The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor-stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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16. Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM.
- Author
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Shen Y, Nakajima M, Ahmad MR, Kojima S, Homma M, and Fukuda T
- Subjects
- Adhesiveness, Humidity, Microscopy, Atomic Force instrumentation, Nanotechnology, Robotics, Surface Properties, Cell Adhesion physiology, Microscopy, Electron, Scanning instrumentation, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae ultrastructure
- Abstract
A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
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17. Study of the time effect on the strength of cell-cell adhesion force by a novel nano-picker.
- Author
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Shen Y, Nakajima M, Kojima S, Homma M, and Fukuda T
- Subjects
- Microscopy, Atomic Force, Surface Properties, Time Factors, Cell Adhesion, Nanoparticles, Robotics methods
- Abstract
Cell's adhesion is important to cell's interaction and activates. In this paper, a novel method for cell-cell adhesion force measurement was proposed by using a nano-picker. The effect of the contact time on the cell-cell adhesion force was studied. The nano-picker was fabricated from an atomic force microscopy (AFM) cantilever by nano fabrication technique. The cell-cell adhesion force was measured based on the deflection of the nano-picker beam. The result suggests that the adhesion force between cells increased with the increasing of contact time at the first few minutes. After that, the force became constant. This measurement methodology was based on the nanorobotic manipulation system inside an environmental scanning electron microscope. It can realize both the observation and manipulation of a single cell at nanoscale. The quantitative and precise cell-cell adhesion force result can be obtained by this method. It would help us to understand the single cell interaction with time and would benefit the research in medical and biological fields potentially., (Copyright © 2011. Published by Elsevier Inc.)
- Published
- 2011
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18. Open-well stability of reagent cartridge for affinity column-mediated immunoassay of blood tacrolimus concentration.
- Author
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Doki K, Homma M, and Kohda Y
- Subjects
- Affinity Labels standards, Humans, Immunoassay methods, Immunoassay standards, Indicators and Reagents standards, Technology, Pharmaceutical methods, Tacrolimus blood, Technology, Pharmaceutical standards
- Published
- 2010
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19. Functional transfer of an essential aspartate for the ion-binding site in the stator proteins of the bacterial flagellar motor.
- Author
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Terashima H, Kojima S, and Homma M
- Subjects
- Aspartic Acid genetics, Bacterial Proteins chemistry, Bacterial Proteins genetics, Binding Sites, Cell Migration Assays, Immunoblotting, Mutagenesis, Site-Directed, Mutation genetics, Protein Binding, Sodium Channels chemistry, Sodium Channels genetics, Vibrio alginolyticus, Aspartic Acid chemistry, Aspartic Acid metabolism, Bacterial Proteins metabolism, Sodium Channels metabolism
- Abstract
Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H(+) or Na(+)) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na(+) across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na(+)-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na(+)-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex., (Copyright 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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20. Effects of chloride ion binding on the photochemical properties of salinibacter sensory rhodopsin I.
- Author
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Suzuki D, Furutani Y, Inoue K, Kikukawa T, Sakai M, Fujii M, Kandori H, Homma M, and Sudo Y
- Subjects
- Amino Acid Sequence, Models, Biological, Models, Molecular, Molecular Sequence Data, Protein Binding, Sequence Alignment, Spectrum Analysis methods, Bacteroidetes enzymology, Chlorides metabolism, Ions metabolism, Sensory Rhodopsins chemistry, Sensory Rhodopsins metabolism
- Abstract
Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl(-)-dependent manner with a K(m) of 307+/-56 mM, showing that Cl(-)-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO(3)), implying that I(-), Br(-), and NO(3)(-) can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl(-)-binding site. In light of these results, we discuss the effects of the Cl(-) binding to SRI and the roles of Cl(-) binding in its function.
- Published
- 2009
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21. Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli.
- Author
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Inoue Y, Lo CJ, Fukuoka H, Takahashi H, Sowa Y, Pilizota T, Wadhams GH, Homma M, Berry RM, and Ishijima A
- Subjects
- Bacterial Outer Membrane Proteins genetics, Escherichia coli chemistry, Vibrio alginolyticus chemistry, Vibrio alginolyticus metabolism, ATP-Binding Cassette Transporters metabolism, Bacterial Outer Membrane Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Flagella metabolism, Molecular Motor Proteins metabolism, Sodium metabolism, Torque
- Abstract
The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na(+)-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na(+)). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at approximately 2200 pN nm) from stall up to a "knee" speed of approximately 420 Hz, and then falls linearly with speed, extrapolating to zero torque at approximately 910 Hz. Motors containing one to five stators generated approximately 200 pN nm per stator at speeds up to approximately 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to approximately 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to approximately 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.
- Published
- 2008
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22. Flagellar motility in bacteria structure and function of flagellar motor.
- Author
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Terashima H, Kojima S, and Homma M
- Subjects
- Movement, Bacterial Physiological Phenomena, Flagella physiology, Flagella ultrastructure
- Abstract
Bacterial flagella are filamentous organelles that drive cell locomotion. They thrust cells in liquids (swimming) or on surfaces (swarming) so that cells can move toward favorable environments. At the base of each flagellum, a reversible rotary motor, which is powered by the proton- or the sodium-motive force, is embedded in the cell envelope. The motor consists of two parts: the rotating part, or rotor, that is connected to the hook and the filament, and the nonrotating part, or stator, that conducts coupling ion and is responsible for energy conversion. Intensive genetic and biochemical studies of the flagellum have been conducted in Salmonella typhimurium and Escherichia coli, and more than 50 gene products are known to be involved in flagellar assembly and function. The energy-coupling mechanism, however, is still not known. In this chapter, we survey our current knowledge of the flagellar system, based mostly on studies from Salmonella, E. coli, and marine species Vibrio alginolyticus, supplemented with distinct aspects of other bacterial species revealed by recent studies.
- Published
- 2008
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23. Rebleeding from ruptured intracranial aneurysms in North Eastern Province of Japan. A cooperative study.
- Author
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Tanno Y, Homma M, Oinuma M, Kodama N, and Ymamoto T
- Subjects
- Aneurysm, Ruptured epidemiology, Cerebral Angiography, Female, Hemorrhage epidemiology, Humans, Imaging, Three-Dimensional, Intracranial Aneurysm epidemiology, Japan epidemiology, Magnetic Resonance Angiography, Male, Recurrence, Retrospective Studies, Time Factors, Trauma Severity Indices, Aneurysm, Ruptured complications, Hemorrhage etiology, Intracranial Aneurysm complications
- Abstract
Object: Rebleeding from ruptured intracranial aneurysms is a major cause of death and disability. With regard to the factors that precipitate the rebleeding and influence the time course after initial bleeding, previous reports differ in their results, and the number of patients investigated was not sufficient for valid conclusions. This study was thus designed to clarify the factors related to rebleeding from ruptured intracranial aneurysms in a large group of patients of the North Eastern Province of Japan., Methods: We found 181 patients with rebleeding after hospitalization among 5612 cases of ruptured intracranial aneurysms from January 1997 to December 2001 in 33 major hospitals in the North Eastern Province of Japan. We analyzed the data with respect to the time course after bleeding and rebleeding, the arterial blood pressure, the situation when rebleeding occurred, the methods of neuroimaging, the level of consciousness, the treatment and the outcome., Results: Of 181 patients who were hospitalized, rebleeding occurred in 65 (35.9%) within 3 h and 88 (48.6%) within 6 h after the initial subarachnoid hemorrhage (SAH). The consciousness level before the rebleeding varied widely in distribution, but belonged to the drowsiness or less [Japan coma scale (JCS) single-digit] in 83 patients (45.8%), but after rebleeding, JCS triple-digits (semicoma to coma) included 152 patients (84.0%). Systolic arterial blood pressure prior to rebleeding was most commonly between 120 and 140 mmHg. Rebleeding did occur more frequently during angiography (totally 29 patients, 20%) and much less frequently during 3D-CTA and MRA procedures (a single case). Treatment consisted of aneurysm neck clipping in 72 patients (40.0%), endovascular therapy with coils in 4 patients (2.2%) and conservative ones in 103 patients (56.9%). As to outcome, 109 patients with rebleeding (60.2%) died in 3 months following initial SAH., Conclusion: Rebleeding occurs more frequently in the earlier period after the initial SAH than previously believed. Thus, more aggressive pharmacologically induced systemic arterial hypotension appears to be important for preventing rebleeding but ultimate outcome of more aggressive hypotension is yet to be determined. If feasible, in order to avoid catheter-angiography related rebleeding, evaluations solely with 3D-CTA and MRA should be in consideration and earlier surgical intervention seems essential as rebleeding does occur often within the first 3 h of onset.
- Published
- 2007
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24. Visualization of functional rotor proteins of the bacterial flagellar motor in the cell membrane.
- Author
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Fukuoka H, Sowa Y, Kojima S, Ishijima A, and Homma M
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Molecular Motor Proteins chemistry, Molecular Motor Proteins genetics, Movement, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Bacterial Proteins metabolism, Cell Membrane metabolism, Flagella metabolism, Molecular Motor Proteins metabolism
- Abstract
The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP-FliG was expressed in a DeltafliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP-FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble.
- Published
- 2007
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25. SDF-1 alpha regulates mesendodermal cell migration during frog gastrulation.
- Author
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Fukui A, Goto T, Kitamoto J, Homma M, and Asashima M
- Subjects
- Animals, Chemokine CXCL12, Chemokines, CXC biosynthesis, Chemokines, CXC genetics, Endoderm metabolism, Gastrula metabolism, Mesoderm metabolism, Receptors, CXCR4 biosynthesis, Receptors, CXCR4 genetics, Xenopus laevis, Cell Movement physiology, Chemokines, CXC physiology, Endoderm cytology, Endoderm physiology, Gastrula cytology, Gastrula physiology, Mesoderm cytology, Mesoderm physiology
- Abstract
During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1alpha expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1alpha caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1alpha-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1alpha, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation.
- Published
- 2007
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26. A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation.
- Author
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Homma M, Inui M, Fukui A, Michiue T, Okabayashi K, and Asashima M
- Subjects
- Activins metabolism, Amino Acid Sequence, Animals, Base Sequence, Body Patterning genetics, Cell Nucleus metabolism, DNA Primers, Galactosides, Gene Expression Profiling, Genetic Vectors, In Situ Hybridization, Indoles, Molecular Sequence Data, Oligonucleotides, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Signal Transduction genetics, T-Box Domain Proteins genetics, Xenopus Proteins genetics, Body Patterning physiology, Gastrula physiology, Gene Expression Regulation, Developmental genetics, Signal Transduction physiology, T-Box Domain Proteins metabolism, Xenopus Proteins metabolism, Xenopus laevis embryology, Xenopus laevis genetics
- Abstract
Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression.
- Published
- 2007
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27. Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes.
- Author
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Yonekura K, Yakushi T, Atsumi T, Maki-Yonekura S, Homma M, and Namba K
- Subjects
- Bacterial Proteins metabolism, Cryoelectron Microscopy, Flagella metabolism, Image Processing, Computer-Assisted, Immunohistochemistry, Lipid Bilayers, Models, Molecular, Molecular Motor Proteins metabolism, Peptidoglycan metabolism, Protein Binding, Protein Conformation, Sodium Channels metabolism, Bacterial Proteins ultrastructure, Flagella ultrastructure, Liposomes, Molecular Motor Proteins ultrastructure, Sodium metabolism, Sodium Channels ultrastructure
- Abstract
A motor protein complex of the bacterial flagellum, PomA/B from Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB, and converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog called MotA/B, which utilizes proton instead of sodium ion. PomB and MotB have a peptidoglycan-binding motif in their C-terminal region, and therefore PomA/B and MotA/B are regarded as the stator. Energy filtering electron cryomicroscopy enhanced the image contrast of the proteins reconstituted into liposomes and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides. Image analysis combined with gold labeling and deletion of the peptidoglycan-binding motif revealed that the longer one, approximately 70 A long, is likely to correspond to the periplasmic domain, and the other, about half size, to the cytoplasmic domain.
- Published
- 2006
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28. Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor.
- Author
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Fukuoka H, Yakushi T, Kusumoto A, and Homma M
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Base Sequence, DNA, Bacterial genetics, Flagella metabolism, Green Fluorescent Proteins chemistry, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Molecular Motor Proteins chemistry, Molecular Motor Proteins genetics, Movement physiology, Protein Structure, Tertiary, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sodium metabolism, Sodium Channels chemistry, Sodium Channels genetics, Subcellular Fractions metabolism, Vibrio alginolyticus genetics, Bacterial Proteins metabolism, Molecular Motor Proteins metabolism, Sodium Channels metabolism, Vibrio alginolyticus metabolism
- Abstract
PomA and PomB are transmembrane proteins that form the stator complex in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed to surround the rotor part of the flagellar motor. We constructed and observed green fluorescent protein (GFP) fusions of the stator proteins PomA and PomB in living cells to clarify how stator proteins are assembled and installed into the flagellar motor. We were able to demonstrate that GFP-PomA and GFP-PomB localized to a cell pole dependent on the presence of the polar flagellum. Localization of the GFP-fused stator proteins required their partner subunit, PomA or PomB, and the C-terminal domain of PomB, which has a peptidoglycan-binding motif. Each of the GFP-fused stator proteins was co-isolated with its partner subunit from detergent-solubilized membrane. From these lines of evidence, we have demonstrated that the stator proteins are incorporated into the flagellar motor as a PomA/PomB complex and are fixed to the cell wall via the C-terminal domain of PomB.
- Published
- 2005
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29. The conserved charged residues of the C-terminal region of FliG, a rotor component of the Na+-driven flagellar motor.
- Author
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Yorimitsu T, Mimaki A, Yakushi T, and Homma M
- Subjects
- Amino Acid Sequence, Cloning, Molecular, Conserved Sequence, Endopeptidases, Escherichia coli metabolism, Green Fluorescent Proteins, Luminescent Proteins metabolism, Membrane Proteins chemistry, Membrane Proteins genetics, Membrane Proteins metabolism, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Sequence Homology, Amino Acid, Subcellular Fractions, Swimming, Vibrio metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Escherichia coli chemistry, Flagella metabolism, Mutation genetics
- Abstract
FliG is an essential component of the flagellar motor and functions in flagellar assembly, torque generation and regulation of the direction of flagellar rotation. The five charged residues important for the rotation of the flagellar motor were identified in Escherichiacoli FliG (FliG(E)). These residues are clustered in the C terminus and are all conserved in FliG(V) of the Na(+)-driven motor of Vibrioalginolyticus (Lys284, Arg301, Asp308, Asp309 and Arg317). To investigate the roles of these charged residues in the Na(+)-driven motor, we cloned the VibriofliG gene and introduced single or multiple substitutions into the corresponding positions in FliG(V). FliG(V) with double Ala replacements in all possible combinations at these five conserved positions still retained significant motile ability, although some of the mutations completely eliminated the function of FliG(E). All of the triple mutants constructed in this study also remained motile. These results suggest that the important charged residues may be located in different places and the conserved charged residues are not so important for the Na(+)-driven flagellar motor of Vibrio. The chimeric FliG protein (FliG(VE)), composed of the N-terminal domain from V.alginolyticus and the C-terminal domain from E.coli, functions in Vibrio cells. The mutations of the charge residues of the C-terminal region in FliG(VE) affected swarming ability as in E.coli. Both the FliG(V) and the FliG(VE) proteins with the triple mutation were more susceptible to proteolysis than proteins without the mutation, suggesting that their conformations were altered.
- Published
- 2003
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30. Torque-speed relationship of the Na+-driven flagellar motor of Vibrio alginolyticus.
- Author
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Sowa Y, Hotta H, Homma M, and Ishijima A
- Subjects
- Culture Media, Flagella physiology, Sodium physiology, Vibrio physiology
- Abstract
The torque-speed relationship of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated. The rotation rate of the motor was measured by following the position of a bead, attached to a flagellar filament, using optical nanometry. In the presence of 50mM NaCl, the generated torque was relatively constant ( approximately 3800pNnm) at lower speeds (speeds up to approximately 300Hz) and then decreased steeply, similar to the H(+)-driven flagellar motor of Escherichia coli. When the external NaCl concentration was varied, the generated torque of the flagellar motor was changed over a wide range of speeds. This result could be reproduced using a simple kinetic model, which takes into consideration the association and dissociation of Na(+) onto the motor. These results imply that for a complete understanding of the mechanism of flagellar rotation it is essential to consider both the electrochemical gradient and the absolute concentration of the coupling ion.
- Published
- 2003
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31. Ion-coupling determinants of Na+-driven and H+-driven flagellar motors.
- Author
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Asai Y, Yakushi T, Kawagishi I, and Homma M
- Subjects
- Amino Acid Sequence, Bacterial Outer Membrane Proteins genetics, Bacterial Outer Membrane Proteins metabolism, Bacterial Proteins genetics, Escherichia coli genetics, Flagella genetics, Hydrogen-Ion Concentration, Membrane Proteins genetics, Membrane Proteins metabolism, Molecular Sequence Data, Plasmids, Sequence Homology, Amino Acid, Sodium Channels genetics, Transformation, Genetic, Bacterial Proteins metabolism, Escherichia coli physiology, Flagella physiology, Hydrogen, Molecular Motor Proteins physiology, Sodium, Sodium Channels metabolism, Vibrio physiology
- Abstract
The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7(E)) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7(E)) supported sodium-driven motility in motA or motB E.coli cells, and the swimming speed was even higher than with the original stator of E.coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7(E)) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor.
- Published
- 2003
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32. The systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na+-driven flagellar motor component PomA.
- Author
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Yorimitsu T, Sowa Y, Ishijima A, Yakushi T, and Homma M
- Subjects
- Amino Acid Sequence, Dose-Response Relationship, Drug, Escherichia coli metabolism, Flagella metabolism, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Structure, Tertiary, Sodium Chloride pharmacology, Vibrio metabolism, Bacterial Outer Membrane Proteins chemistry, Cytoplasm metabolism, Sodium metabolism
- Abstract
PomA, a homolog of MotA in the H+-driven flagellar motor, is an essential component for torque generation in the Na+-driven flagellar motor. Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process. These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively. To explore the role of these charged residues in the Na+-driven motor, we replaced them with other amino acids. However, unlike in the H+-driven motor, both of the single and the double PomA mutants were functional. Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized. Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The swimming speed of the AAQQQ strain was as fast as that of the wild-type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased. We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized. The charged residues at positions 88-99 of PomA may not be essential for torque generation in the Na+-driven motor and might play a role in motor function different from that of the equivalent residues of the H+-driven motor.
- Published
- 2002
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33. MRNA and enzyme activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 are elevated in C57BL/KsJ-db/db mice.
- Author
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Aoki K, Homma M, Hirano T, Oka K, Satoh S, Mukasa K, Ito S, and Sekihara H
- Subjects
- 11-beta-Hydroxysteroid Dehydrogenase Type 1, Animals, Base Sequence, Blood Glucose analysis, Body Weight, Corticosterone blood, DNA Primers, Hydroxysteroid Dehydrogenases genetics, Insulin blood, Male, Mice, Mice, Inbred C57BL, Organ Size, Hydroxysteroid Dehydrogenases metabolism, Liver enzymology, RNA, Messenger metabolism
- Abstract
To evaluate the importance of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in insulin resistant diabetic C57BL/KsJ-db/db mice, we measured the activity and mRNA level of 11beta-HSD1 in the liver of db/db mice and their heterozygote litter mates, db/+m mice. The blood glucose, plasma insulin, and corticosterone levels of db/db mice were significantly higher than those of db/+m mice. Despite hyperinsulinemia, the activity level of this enzyme was significantly higher in db/db mice, and the mRNA level of hepatic 11beta-HSD1 was also significantly higher in db/db mice. Since hepatic 11beta-HSD1 in vivo mainly functions as 11-keto-reductase and does not work as 11beta-oxidase, these results suggest that the rate of hepatic conversion of 11-dehydrocorticosterone to corticosterone is increased in db/db mice, resulting in higher glucocorticoid activity in the liver. The increased hepatic corticosterone concentration due to the elevation of 11beta-HSD1 and high plasma corticosterone concentration may antagonize the action of insulin and cause insulin resistance. These findings have a potentially important implication for relationships between increased hepatic 11beta-HSD1 and insulin resistance in db/db mice. The present paper is the first to demonstrate the increased activities and mRNA level of hepatic 11beta-HSD1 in db/db mice.
- Published
- 2001
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34. Inversion of thermosensing property of the bacterial receptor Tar by mutations in the second transmembrane region.
- Author
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Nishiyama S, Maruyama IN, Homma M, and Kawagishi I
- Subjects
- Amino Acid Sequence, Aspartic Acid pharmacology, Bacterial Proteins chemistry, Bacterial Proteins genetics, Chemoreceptor Cells, Chemotaxis drug effects, Chemotaxis physiology, Cold Temperature, Escherichia coli drug effects, Escherichia coli genetics, Glycerol pharmacology, Hot Temperature, Membrane Proteins chemistry, Membrane Proteins genetics, Methylation, Molecular Sequence Data, Phenotype, Protein Structure, Secondary, Receptors, Cell Surface chemistry, Receptors, Cell Surface genetics, Receptors, Cell Surface physiology, Signal Transduction drug effects, Temperature, Bacterial Proteins physiology, Escherichia coli physiology, Escherichia coli Proteins, Membrane Proteins physiology, Mutation
- Abstract
The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate alpha-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190-210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar., (Copyright 1999 Academic Press.)
- Published
- 1999
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35. Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations in putative channel components.
- Author
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Kojima S, Asai Y, Atsumi T, Kawagishi I, and Homma M
- Subjects
- Amiloride pharmacology, Amino Acid Sequence, Bacterial Proteins, Base Sequence, Binding Sites genetics, DNA Primers genetics, Drug Resistance, Microbial genetics, Genes, Bacterial, Models, Molecular, Molecular Motor Proteins physiology, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Protein Conformation, Sodium Channels drug effects, Sodium Channels genetics, Sodium Channels physiology, Vibrio drug effects, Vibrio genetics, Vibrio physiology, Amiloride analogs & derivatives, Flagella drug effects, Flagella physiology, Molecular Motor Proteins drug effects, Molecular Motor Proteins genetics, Sodium metabolism
- Abstract
The rotation of the Na+-driven flagellar motor is specifically and strongly inhibited by phenamil, an amiloride analog. Here, we provide the first evidence that phenamil interacts directly with the Na+-channel components (PomA and PomB) of the motor. The alterations in Mpar (motility resistant to phenamil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pomB from two Mpar strains, NMB205 and NMB201, and found a substitution in pomA (Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Both residues are predicted to be near the cytoplasmic ends of the putative transmembrane segments. Mutational analyses at PomA-Asp148 and PomB-Pro16 suggest that a certain structural change around these residues affects the sensitivity of the motor to phenamil. Co-expression of the PomA D148Y and PomB P16S proteins resulted in an Mpar phenotype which seemed to be less sensitive to phenamil than either of the single mutants, although motility was more severely impaired in the absence of inhibitors. These results support the idea that PomA and PomB interact with each other and suggest that multiple residues, including Asp148 of PomA and Pro16 of PomB, constitute a high-affinity phenamil-binding site at the inner face of the PomA/PomB channel complex., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
36. Extrashot-ODS, a syringe-type minicolumn sample injector for a reversed-phase high-performance liquid chromatographic column. Application to antiepileptics in human sera.
- Author
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Kouno Y, Ishikura C, Homma M, and Oka K
- Subjects
- Anticonvulsants administration & dosage, Anticonvulsants therapeutic use, Carbamazepine administration & dosage, Carbamazepine blood, Carbamazepine therapeutic use, Drug Therapy, Combination, Epilepsy blood, Epilepsy drug therapy, Humans, Immunoenzyme Techniques, Phenobarbital administration & dosage, Phenobarbital blood, Phenobarbital therapeutic use, Phenytoin administration & dosage, Phenytoin blood, Phenytoin therapeutic use, Anticonvulsants blood, Chromatography, High Pressure Liquid instrumentation
- Abstract
Extrashot-ODS (EXS-ODS) is a syringe-type minicolumn developed for sample injection into reversed-phase high-performance liquid chromatographic columns. EXS-ODS consists of (a) a stainless-steel needle fitted to an ordinary syringe-loading sample injector for HPLC, (b) a 45-microl minicolumn tube made of polytetrafluoroethylene (PTFE) and packed with ODS-silica and (c) a minicolumn holder made of polystyrene, which is connected to the needle on one side and the other side is shaped so as to be fitted with a solvent syringe. Using the device, we simultaneously analyzed three antiepileptics in 20 microl of human sera. First, we introduced a 20-microl serum specimen diluted with 100 microl of buffer solution into the device and, second, 100 microl of distilled water. Then the device was attached to the HPLC injector and 130 microl of methanol were introduced into the HPLC column through the device. Then, reversed-phase HPLC was conducted in the usual manner, with the chromatogram reading at a wavelength of 210 nm for the assays of 5,5-diphenylhydantoin, phenobarbital and carbamazepine. The results obtained by direct peak-height calibration were comparable to those given by the immunological method.
- Published
- 1997
- Full Text
- View/download PDF
37. Chromatographic identification of phenolic compounds in human urine following oral administration of the herbal medicines Daisaiko-to and Shosaiko-to.
- Author
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Li C, Homma M, and Oka K
- Subjects
- Chromatography, High Pressure Liquid, Humans, Plant Extracts pharmacokinetics, Spectrophotometry, Ultraviolet, Anthraquinones urine, Drugs, Chinese Herbal pharmacokinetics, Flavonoids urine
- Abstract
Chemical identification of the compounds in human urine following administration of the traditional Chinese medicines, Daisaiko-to and Shosaiko-to (Dachaihu-tang and Xiaochaihu-tang in Chinese, respectively), was achieved by using a linear relationship between the logarithm of the capacity factor, log k', and that of the volume fraction of CH3CN, log X(s)(vol), in the aqueous mobile phase: -log k'=A+B log X(s)(vol). Comparison of the slope, B, and the intercept, A, between the urinary compound and its suspected authentic specimen gave satisfactory results in the chemical identification. We applied this method to the initial stage of pharmacokinetic studies on the herbal medicines and identified seven flavonoids and two anthraquinone derivatives in the urine specimens obtained after herbal administration.
- Published
- 1997
- Full Text
- View/download PDF
38. Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor.
- Author
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Kojima S, Atsumi T, Muramoto K, Kudo S, Kawagishi I, and Homma M
- Subjects
- Amiloride pharmacology, Drug Resistance, Microbial genetics, Flagella metabolism, Flagella physiology, Vibrio drug effects, Amiloride analogs & derivatives, Flagella drug effects, Mutation, Sodium metabolism, Vibrio genetics
- Abstract
The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil. The motility of the wild-type (Mpa(s)) was inhibited by 50 microM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 microM phenamil. The Ki value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain. The rotation rate of the wild-type Na+-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpas motors. The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.
- Published
- 1997
- Full Text
- View/download PDF
39. Rotational fluctuation of the sodium-driven flagellar motor of Vibrio alginolyticus induced by binding of inhibitors.
- Author
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Muramoto K, Magariyama Y, Homma M, Kawagishi I, Sugiyama S, Imae Y, and Kudo S
- Subjects
- Amiloride analogs & derivatives, Amiloride pharmacology, Cell Division drug effects, Computer Simulation, Flagella metabolism, Microscopy, Models, Biological, Molecular Structure, Movement drug effects, Protein Binding, Sodium antagonists & inhibitors, Sodium Channel Blockers, Sodium Channels drug effects, Vibrio growth & development, Vibrio metabolism, Flagella physiology, Sodium metabolism, Vibrio physiology
- Abstract
Rotation of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated under the influence of inhibitors specific to the motor, amiloride and phenamil. The rotation rate of a single flagellum on a cell stuck to a glass slide was examined using laser dark-field microscopy. In the presence of 50 mM NaCl, the average rotation rate (omega) was about 600 r.p.s. with a standard deviation (sigma omega) of 9% of omega. When omega was decreased to about 200 r.p.s. by the presence of 1.5 mM amiloride, sigma omega increased to 15% of omega. On the other hand, when omega was decreased to about 200 r.p.s. by the addition of 0.6 microM phenamil, a large increase in sigma omega up to 50% of omega, was observed. Similarly large fluctuations were observed at other concentrations of phenamil. These observations suggest that dissociation of phenamil from the motor was much slower than that of amiloride. A very low concentration of phenamil caused a transient but substantial reduction in rotation rate. This might suggest that binding of only a single molecule of phenamil strongly inhibits the torque generation in the flagellar motor.
- Published
- 1996
- Full Text
- View/download PDF
40. Sequential activation of MAP kinase cascade by angiotensin II in opossum kidney cells.
- Author
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Terada Y, Tomita K, Homma MK, Nonoguchi H, Yang T, Yamada T, Yuasa Y, Krebs EG, and Marumo F
- Subjects
- Animals, Calcium-Calmodulin-Dependent Protein Kinases drug effects, Cells, Cultured, Kidney Tubules, Proximal drug effects, Opossums, Phosphorylation drug effects, Angiotensin II pharmacology, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Kidney Tubules, Proximal enzymology
- Abstract
Angiotensin II (Ang II) is a potent regulator of proximal tubule functions, including transport, metabolism, and cell proliferation. The opossum kidney (OK) cell line is a useful model of renal proximal tubule. Mitogen-activated protein (MAP) kinases are rapidly phosphorylated and activated in response to various agonists. We investigated Ang II effects on serine/threonine kinase cascades in OK cells. The major findings of the present study are that Ang II stimulated MAP kinase kinase (MAPKK), MAP kinase (MAPK), and S6 kinase activities, and that it increased phosphorylation of Raf-1 kinase and p42 MAP kinase in OK cells. These stimulations of kinases were dose-dependent (from 10(-6) to 10(-11) M). The time course of activation was sequential; the peak stimulation was reached at 5 to 10 minutes for Raf-1 kinase, MAPKK and MAPK, and at 20 minutes for S6 kinase. The activation of MAPK was inhibited by approximately 70% with prolonged 24-hour PMA pretreatment or in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein and herbimycin) did not inhibit AngII-induced MAPK activity. This activation of MAPK was also inhibited via AT1 receptor antagonist, Dup753 and pertussis toxin. This evidence suggests that the activation of serine/threonine cascades by Ang II is largely dependent on PMA-sensitive PKC, and is not dependent on tyrosine kinase and pertussis toxin.
- Published
- 1995
- Full Text
- View/download PDF
41. AVP inhibits EGF-stimulated MAP kinase cascade in Madin-Darby canine kidney cells.
- Author
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Yamada T, Terada Y, Homma MK, Nonoguchi H, Sasaki S, Yuasa Y, Tomita K, and Marumo F
- Subjects
- Animals, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Cells, Cultured, Dogs, Dose-Response Relationship, Drug, Genistein, Isoflavones pharmacology, Kidney Tubules, Distal enzymology, Mitogen-Activated Protein Kinase Kinases, Phosphorylation, Protein Kinases drug effects, Protein Kinases metabolism, Protein Serine-Threonine Kinases drug effects, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins drug effects, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-raf, Ribosomal Protein S6 Kinases, Thymidine metabolism, Arginine Vasopressin pharmacology, Calcium-Calmodulin-Dependent Protein Kinases drug effects, Epidermal Growth Factor pharmacology, Kidney Tubules, Distal drug effects
- Abstract
We investigated the effects of epidermal growth factor (EGF) and arginine vasopressin (AVP) on Raf-1-MAP kinase cascade, including Raf-1-kinase (Raf-1-K), MAP kinase kinase (MAPKK), MAP kinase (MAPK) and S6 kinase (S6K) in Madin-Darby canine kidney (MDCK) cells. In a dose-dependent manner (10(-10) M to 10(-6) M), EGF increased autophosphorylation of Raf-1-K and activated MAPKK, MAPK and S6K. Sequential activation of these kinases was indicated by their peak times of activation (Raf-1-K 5 min; MAPKK 10 min; MAPK 15 min; and S6K 30 min). AVP (10(-9) M to 10(-6) M) inhibited EGF-stimulated MAP kinase cascade. 8-Bromo-cyclic AMP (cAMP) could mimic the inhibitory effect of AVP on EGF-stimulated MAP kinase cascade. These results were confirmed using H-89, an inhibitor of protein kinase A (PKA) that blocked the effect of AVP on EGF-stimulated MAPK activity. We conclude that AVP inhibits EGF-stimulated Raf-1-K, MAPKK, MAPK, and S6K activity via cAMP in MDCK cells. Our results indicate that MAP kinase cascade may play an important role in integrating the effects of AVP and EGF on distal tubule function.
- Published
- 1995
- Full Text
- View/download PDF
42. High-speed rotation and speed stability of the sodium-driven flagellar motor in Vibrio alginolyticus.
- Author
-
Muramoto K, Kawagishi I, Kudo S, Magariyama Y, Imae Y, and Homma M
- Subjects
- Biophysical Phenomena, Biophysics, Carbonyl Cyanide m-Chlorophenyl Hydrazone pharmacology, Cations chemistry, Cations metabolism, Cell Movement drug effects, Cell Movement physiology, Flagella metabolism, Lasers, Membrane Potentials, Microscopy, Confocal methods, Sodium chemistry, Vibrio drug effects, Flagella chemistry, Flagella physiology, Rotation, Sodium pharmacology, Vibrio metabolism
- Abstract
The Na(+)-driven flagellar motor in Vibrio alginolyticus rotates very fast. Rotation of a single flagellum on a stuck cell was measured by laser darkfield microscopy with submillisecond temporal resolution. The rotation rate increased with increasing external concentration of NaCl, and reached 1000 r.p.s. at 300 mM NaCl. The Na+ influx through the motor should determine the rotation period (tau) and affect the speed stability. Fluctuation of the rotation period was analyzed at various rotation rates (from approximately 50 r.p.s. to approximately 1000 r.p.s.), which were changed by changing the external concentration of NaCl and the addition of a protonophore or a specific inhibitor. At high rotation rates (over 400 r.p.s.), the observed rotation was stable, and the standard deviation of tau (sigma tau) ranged from 7% to 16% of the average rotation period (< tau >). At low rotation rates (under 100 r.p.s), the rotation period tended to fluctuate, and the distributions of tau were non-Gaussian. The value of sigma tau ranged from 10 to 30% of < tau >. However, the observed minimum value of sigma tau at various rotation rates was approximately equal to the calculated standard deviation due to the rotational diffusion of the flagellar filament. These results suggest that the torque was stably generated at various Na+ influxes through the motor. We observed large fluctuations that cannot be explained by rotational diffusion. We discuss the factors that induce the large fluctuation.
- Published
- 1995
- Full Text
- View/download PDF
43. Simple and accurate high-performance liquid chromatographic method for the measurement of three antiepileptics in therapeutic drug monitoring.
- Author
-
Kouno Y, Ishikura C, Homma M, and Oka K
- Subjects
- Carbamazepine blood, Chromatography, High Pressure Liquid methods, Humans, Immunoenzyme Techniques, Phenobarbital blood, Phenytoin blood, Anticonvulsants blood, Drug Monitoring methods
- Abstract
A high-performance liquid chromatographic method for simultaneous determination of three antiepileptics, phenytoin, phenobarbital, and carbamazepine, in serum for therapeutic drug monitoring is described. The drugs were extracted and injected onto a silica-gel column using a syringe-type minicolumn, Extrashot-Silica, packed with diatomaceous earth granules. We used dichloromethane for extraction-injection and n-hexane containing 0.2% acetic acid, 2% ethanol, and 15% dichloromethane for the mobile phase of a silica-gel HPLC. The eluent was monitored with a UV detector set at 240 nm. Linear relationships between the amount of drug and peak height were confirmed at 1-20 micrograms/ml in serum for carbamazepine and 5-40 micrograms/ml in serum for phenytoin and phenobarbital. When a 5-microliters aliquot of serum was subjected to this method, the observed detection limits of the drugs were far less than therapeutic concentrations. Thus, our method was simple and accurate enough to be used in routine therapeutic drug monitoring and basic pharmacokinetic studies.
- Published
- 1993
- Full Text
- View/download PDF
44. Low-birthweight hypertension via enzyme and receptor imprintings of glucocorticoid.
- Author
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Oka K, Hirano T, and Homma M
- Subjects
- 11-beta-Hydroxysteroid Dehydrogenases, Adult, Female, Fetus physiology, Humans, Hypertension genetics, Pregnancy, Prenatal Exposure Delayed Effects, Hydroxysteroid Dehydrogenases genetics, Hypertension enzymology, Receptors, Glucocorticoid genetics
- Published
- 1993
- Full Text
- View/download PDF
45. Pharmacokinetic evaluation of traditional Chinese herbal remedies.
- Author
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Homma M, Oka K, Niitsuma T, and Itoh H
- Subjects
- Histamine H1 Antagonists pharmacokinetics, Humans, Drugs, Chinese Herbal pharmacokinetics, Medicine, Kampo
- Published
- 1993
- Full Text
- View/download PDF
46. Genomic structure of the ME491/CD63 antigen gene and functional analysis of the 5'-flanking regulatory sequences.
- Author
-
Hotta H, Miyamoto H, Hara I, Takahashi N, and Homma M
- Subjects
- Amino Acid Sequence, Base Sequence, Chloramphenicol O-Acetyltransferase biosynthesis, DNA Mutational Analysis, Exons genetics, Humans, Introns genetics, Molecular Sequence Data, Promoter Regions, Genetic genetics, RNA, Messenger genetics, Recombinant Proteins biosynthesis, Tetraspanin 30, Antigens, CD genetics, Platelet Membrane Glycoproteins genetics, Regulatory Sequences, Nucleic Acid genetics
- Abstract
Genomic structure of the ME491/CD63 antigen gene and promoter activity of the 5'-flanking regulatory sequences were studied. The antigen gene consists of eight exons that span 4 kilobase-pairs. Primer extension analysis with RNA from cultured human cells identified three major transcription initiation sites. The 5'-flanking region of exon 1 has features characteristic to promoters of many house-keeping genes and growth-regulating genes. The region is highly GC rich and contains potential binding sites for transcription factors such as Sp1 and ETF, but not a TATA box. The 5'-flanking sequence exerted strong promoter activity when linked to a reporter gene. Deletion mutant analysis of the 5'-flanking sequence has strongly suggested that a potential binding site for AP-1 plays an important role in positively regulating the gene expression.
- Published
- 1992
- Full Text
- View/download PDF
47. A strategy for discovering biologically active compounds with high probability in traditional Chinese herb remedies: an application of saiboku-to in bronchial asthma.
- Author
-
Homma M, Oka K, Yamada T, Niitsuma T, Ihto H, and Takahashi N
- Subjects
- Administration, Oral, Adult, Aged, Asthma metabolism, Chemical Fractionation, Chromatography, High Pressure Liquid, Drugs, Chinese Herbal chemistry, Drugs, Chinese Herbal therapeutic use, Histamine H1 Antagonists therapeutic use, Histamine H1 Antagonists urine, Humans, Male, Mass Spectrometry, Middle Aged, Asthma drug therapy, Drugs, Chinese Herbal pharmacokinetics, Histamine H1 Antagonists pharmacokinetics, Medicine, Kampo
- Abstract
A novel strategy for discovering biologically active components in traditional Chinese herb remedies was performed from a pharmacokinetic view. The hypothesis was that the active compounds should appear in blood and urine with appropriate blood concentrations and urinary excretion rates after the administration of herbal-extract mixtures. In this research, we applied our procedures to Saiboku-To, one of the most popular Chinese herbal medicines in Japan. Consisting of 10 different plant extracts, it is used for the treatment of bronchial asthma. The analytical method adopted was a rapid-flow fractionation (RFF) for extraction-fractionation of lipophilic components in urine followed by silica-gel high-performance liquid chromatography (HPLC) equipped with a multichannel ultraviolet (uv) absorption detector. beta-D-Glucuronidase-treated urine samples collected before and after the administration of Saiboku-To to healthy and asthmatic subjects were treated with the RFF apparatus to afford three pH-dependent fractions: strongly acidic (S), weakly acidic (W), and neutral (N). HPLC of these fractions, monitored by the multichannel uv detector, showed three new peaks in the postadministrative urine: one in the N fraction, two in the W fraction, and none in the S fraction. A compound in the N fraction was identified with authentic magnolol, a major component in Magnolia officinalis. Two compounds in the W fraction were identified by comparison with authentic samples as 8,9-dihydroxydihydromagnolol and liquiritigenin, metabolites previously isolated from M. officinalis and Glycyrrhiza glabra, respectively.
- Published
- 1992
- Full Text
- View/download PDF
48. Simple and accurate determination of methylpyrazines in biofluids using high-performance liquid chromatography.
- Author
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Kanai M, Kouno Y, Homma M, Yamada K, Oka K, Noguchi M, Abe M, Sakakura K, and Iwata Y
- Subjects
- Animals, Chromatography, High Pressure Liquid, Injections, Intraperitoneal, Male, Models, Biological, Pyrazines blood, Pyrazines urine, Rats, Rats, Inbred Strains, Spectrophotometry, Ultraviolet, Pyrazines analysis
- Abstract
The determination of six methylpyrazines was performed using high-performance liquid chromatography (HPLC). Methylpyrazines were simultaneously extracted and injected onto a silica gel column with a syringe-type minicolumn packed with diatomaceous earth granules. The extraction-injection solvent used was dichloromethane and the mobile phase solvent for HPLC was dichloromethane containing 0.08% of 1.65 M ammonia solution and 0.5% of methanol. Methylpyrazines were detected using an ultraviolet detector set at 275 nm. Linear relationships between the amount of sample and peak height were confirmed from 50 ng/ml to 10 micrograms/ml of the biofluids. When an aliquot of 10 microliters of biofluid was introduced to the minicolumn, the detection limit of methylpyrazines was as low as 30 ng/ml with each pyrazine derivative. The method is simple and accurate and is thus applicable to pharmacokinetic studies which are performed on animals. The results showed that the possible pharmacological effects of methylpyrazines might be evaluated pharmacokinetically using this newly developed technique.
- Published
- 1991
- Full Text
- View/download PDF
49. Ion-sparing diuresis by 2,3-dibenzylbutane-1,4-diol, a synthetic mammalian-lignan derivative.
- Author
-
Hirano T, Homma M, Oka K, Naito T, Hosaka K, and Mitsuhashi H
- Subjects
- Animals, Chlorides blood, Chlorides urine, Furans pharmacology, Furosemide pharmacology, Ion Exchange, Male, Mice, Mice, Inbred BALB C, Natriuresis drug effects, Potassium blood, Potassium urine, Rats, Rats, Inbred Strains, Sodium blood, Benzyl Compounds pharmacology, Diuretics pharmacology, Lignans
- Abstract
Diuretic properties of a synthetic lignan, 2,3-dibenzylbutane-1,4-diol (hattalin), and a naturally occurring arctigenin were examined in BALB/c male mice and Wistar male rats. Intra peritoneal administration of hattalin (50 mg/kg) in mice increased urine volume by 1.7-3.1 fold that of placebo-treated animals 40-260 min after administration (p less than 0.05 vs control). In contrast, 100 mg/kg of arctigenin had no effect on urine volume in mice. Hattalin (100 mg/kg), arctigenin (100 mg/kg), or furosemide (50 mg/kg) as a positive control was administered orally to rats, and accumulated urine volume was measured for up to 6-12 h. The urine volume of animals administered with hattalin showed 1.4-1.5 fold that of placebo-treated animals after 2-6 h of administration (P less than 0.05, n = 10). On the other hand, arctigenin showed no significant effect on urine volume for up to 12 h after administration (n = 8). The urine volume in animals administered with furosemide (n = 10) was 2.0-3.0 fold that of placebo-treated animals (P less than 0.01). Furosemide increased total Na+, K+, or Cl- excretion by 1.9, 1.8 or 2.2 fold, respectively, when compared with placebo-treated controls (P less than 0.01), whereas hattalin decreased Na+ excretion by 3.6 times (P less than 0.01), K+ excretion by 1.4 times (not significant), and Cl- excretion by 3.1 times (P less than 0.01). Serum Na+ and K+ levels did not change in both furosemide- and hattalin-administered rats, however, serum Cl- levels in these animals significantly decreased (P less than 0.01) when compared with controls. The results suggest that the diuretic property of hattalin is due to a novel mechanism which is different from that of furosemide or other diuretics modifying the ion-exchange at the uriniferous tubules.
- Published
- 1991
- Full Text
- View/download PDF
50. Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum.
- Author
-
Homma M, DeRosier DJ, and Macnab RM
- Subjects
- Amino Acid Sequence, Amino Acids analysis, Base Sequence, DNA, Bacterial genetics, Flagella ultrastructure, Genes, Bacterial, Molecular Sequence Data, Repetitive Sequences, Nucleic Acid, Sequence Homology, Nucleic Acid, Bacterial Proteins genetics, Flagella metabolism, Salmonella typhimurium genetics
- Abstract
Within the bacterial flagellum the basal-body rod, the hook, the hook-associated proteins (HAPs), and the helical filament constitute an axial substructure whose elements share structural features and a common export pathway. We present here the amino acid sequences of the hook protein and the three HAPs of Salmonella typhimurium, as deduced from the DNA sequences of their structural genes (flgE, flgK, flgL and fliD, respectively). We compared these sequences with each other and with those for the filament protein (flagellin) and four rod proteins, which have been described previously (Joys, 1985; Homma et al., 1990; Smith & Selander, 1990). Hook protein most strongly resembled the distal rod protein (FlgG) and the proximal HAP (HAP1), which are thought to be attached to the proximal and distal ends of the hook, respectively; the similarities were most pronounced near the N and C termini. Hook protein and flagellin, which occupy virtually identical helical lattices, did not resemble each other strongly but showed some limited similarities near their termini. HAP3 and HAP2, which form the proximal and distal boundaries of the filament, showed few similarities to flagellin, each other, or the other axial proteins. With the exceptions of the N-terminal region of HAP2, and the C-terminal region of flagellin, proline residues were absent from the terminal regions of the axial proteins. Moreover, with the exception of the N-terminal region of HAP2, the terminal regions contained hydrophobic residues at intervals of seven residues. Together, these observations suggest that the axial proteins may have amphipathic alpha-helical structure at their N and C termini. In the case of the filament and the hook, the terminal regions are believed to be responsible for the quaternary interactions between subunits. We suggest that this is likely to be true of the other axial structures as well, and specifically that interaction between N-terminal and C-terminal alpha-helices may be important in the formation of the axial structures of the flagellum. Although consensus sequences were noted among some of the proteins, such as the rod, hook and HAP1, no consensus extended to the entire set of axial proteins. Thus the basis for recognition of a protein for export by the flagellum-specific pathway remains to be identified.
- Published
- 1990
- Full Text
- View/download PDF
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