109 results on '"Kimiko Murakami-Murofushi"'
Search Results
2. 2-Carba-lysophosphatidic acid is a novel β-lysophosphatidic acid analogue with high potential for lysophosphatidic acid receptor activation and autotaxin inhibition
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Keisuke Yoshikawa, Keiko Fukasawa, Kensuke Iwasa, Junken Aoki, Shinji Yamamoto, Akiharu Uwamizu, Takatsugu Hirokawa, Yoshibumi Shimizu, Kimiko Murakami-Murofushi, Mari Gotoh, and Masaki Ishikawa
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Science ,Phospholipid ,Article ,chemistry.chemical_compound ,Lysophosphatidic acid ,Humans ,heterocyclic compounds ,Phosphorylation ,Receptors, Lysophosphatidic Acid ,Multidisciplinary ,Kinase ,Phosphoric Diester Hydrolases ,Biological activity ,Phosphatidic acid ,Transforming Growth Factor alpha ,Lipids ,In vitro ,Recombinant Proteins ,Molecular Docking Simulation ,Alcohol Oxidoreductases ,HEK293 Cells ,chemistry ,Biochemistry ,cardiovascular system ,Solvents ,Medicine ,lipids (amino acids, peptides, and proteins) ,Autotaxin ,biological phenomena, cell phenomena, and immunity ,Lysophospholipids ,Transforming growth factor ,Cell signalling ,HeLa Cells ,Signal Transduction - Abstract
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that, along with its chemically stabilized analogue 2-carba-cyclic phosphatidic acid (2ccPA), induces various biological activities in vitro and in vivo. Although cPA is similar to lysophosphatidic acid (LPA) in structure and synthetic pathway, some of cPA biological functions apparently differ from those reported for LPA. We previously investigated the pharmacokinetic profile of 2ccPA, which was found to be rapidly degraded, especially in acidic conditions, yielding an unidentified compound. Thus, not only cPA but also its degradation compound may contribute to the biological activity of cPA, at least for 2ccPA. In this study, we determined the structure and examined the biological activities of 2-carba-lysophosphatidic acid (2carbaLPA) as a 2ccPA degradation compound, which is a type of β-LPA analogue. Similar to LPA and cPA, 2carbaLPA induced the phosphorylation of the extracellular signal-regulated kinase and showed potent agonism for all known LPA receptors (LPA1–6) in the transforming growth factor-α (TGFα) shedding assay, in particular for LPA3 and LPA4. 2carbaLPA inhibited the lysophospholipase D activity of autotaxin (ATX) in vitro similar to other cPA analogues, such as 2ccPA, 3-carba-cPA, and 3-carba-LPA (α-LPA analogue). Our study shows that 2carbaLPA is a novel β-LPA analogue with high potential for the activation of some LPA receptors and ATX inhibition.
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- 2021
3. Saturated fatty acid is a principal cause of anxiety-like behavior in diet-induced obese rats in relation to serum lysophosphatidyl choline level
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Hiroshi Kunugi, Keiko Fukasawa, Shingo Nakajima, Mari Gotoh, and Kimiko Murakami-Murofushi
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Male ,Sucrose ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Medicine (miscellaneous) ,Adipose tissue ,030209 endocrinology & metabolism ,Anxiety ,Diet, High-Fat ,Rats, Sprague-Dawley ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Internal medicine ,medicine ,Animals ,Obesity ,030212 general & internal medicine ,Neurotransmitter ,Nutrition and Dietetics ,Behavior, Animal ,Chemistry ,Insulin ,Leptin ,Fatty Acids ,Glutamate receptor ,Lysophosphatidylcholines ,Eicosapentaenoic acid ,Rats ,Disease Models, Animal ,Endocrinology ,Saturated fatty acid ,Diet-induced obese - Abstract
Obesity is considered to be a risk factor for neurodegenerative- and psychiatric- diseases including Alzheimer’s disease, schizophrenia, and depression. A high-lard diet is widely used to induce obesity in model animal experiments, which also leads to anxiety-like and depression-like behaviors. However, the contribution of dietary fat source to these abnormal behaviors in obesity is largely unknown. Sprague-Dawley rats were treated with different types of high-fat (lard and olive oil) diet with high sucrose for more than 8 weeks. Anxiety-like behavior (open-field and social interaction tests) and cognitive function (Y-maze test) after the treatment were analyzed. The expression of mRNA related to neurotransmitter and nutrient transporters in the prefrontal cortex were determined using real-time PCR. Serum lipid species were determined using liquid chromatography with tandem mass spectrometry. Both high-fat/high-sucrose diets increased body weight (BW), adipose tissue, and serum leptin level. However, the high-lard/high-sucrose (HL/HS), but not high-olive oil/HS, diet induced anxiety-like behavior in open field and social interaction tests. BW and endocrine hormones such as leptin and insulin were not correlated to anxiety-like behavior. HL/HS diet induced an increase in glutamate transporter and a decrease of glutamate receptor mRNA expressions in the prefrontal cortex. Further, serum lysophosphatidyl choline conjugated with several fatty acids was decreased by HL/HS diet. LPC conjugated with eicosapentaenoic acid (EPA) was strongly correlated with anxiety-like behavior. These results suggest that lipid composition, rather than obesity per se, is a major cause of anxiety-like behavior in high-fat diet-induced obesity. Decreased levels of peripheral LPC conjugated with EPA and altered glutamate system in the prefrontal cortex might be involve in the pathophysiology of the behavioral change.
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- 2019
4. The neuroprotective function of 2-carba-cyclic phosphatidic acid: Implications for tenascin-C via astrocytes in traumatic brain injury
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Hiroko Ikeshima-Kataoka, Yasunori Miyamoto, Misaki Endo, Kimiko Murakami-Murofushi, Kei Hashimoto, Mari Gotoh, and Mari Nakashima
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Lipopolysaccharides ,Traumatic brain injury ,Immunology ,Phosphatidic Acids ,Apoptosis ,Wounds, Stab ,Neuroprotection ,chemistry.chemical_compound ,Mice ,Brain Injuries, Traumatic ,Glial Fibrillary Acidic Protein ,medicine ,Immunology and Allergy ,Animals ,RNA, Messenger ,Cells, Cultured ,Cerebral Cortex ,Neurons ,Gene knockdown ,Mice, Inbred ICR ,biology ,Tenascin C ,Tenascin ,Phosphatidic acid ,Lipid signaling ,medicine.disease ,Cell biology ,medicine.anatomical_structure ,Neuroprotective Agents ,Neurology ,chemistry ,Astrocytes ,Culture Media, Conditioned ,biology.protein ,Female ,RNA Interference ,Neurology (clinical) ,Injections, Intraperitoneal ,Astrocyte - Abstract
We examined the mechanism how 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, regulates neuronal apoptosis in traumatic brain injury (TBI). First, we found 2ccPA suppressed neuronal apoptosis after the injury, and increased the immunoreactivity of tenascin-C (TN-C), an extracellular matrix protein by 2ccPA in the vicinity of the wound region. 2ccPA increased the mRNA expression levels of Tnc in primary cultured astrocytes, and the conditioned medium of 2ccPA-treated astrocytes suppressed the apoptosis of cortical neurons. The neuroprotective effect of TN-C was abolished by knockdown of TN-C. These results indicate that 2ccPA contributes to neuroprotection via TN-C from astrocytes in TBI.
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- 2021
5. Adenine nucleotide translocase 2, a putative target protein for 2-carba cyclic phosphatidic acid in microglial cells
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Satoshi Sakamoto, Tamotsu Tsukahara, Ryoko Tsukahara, Yasuka Sahara, Hiroshi Handa, Mari Gotoh, Nigel Ribeiro, and Kimiko Murakami-Murofushi
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0301 basic medicine ,Phosphatidic Acids ,Apoptosis ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,medicine ,Animals ,Phenylarsine oxide ,Cell Proliferation ,chemistry.chemical_classification ,Reactive oxygen species ,Microglia ,Cell Biology ,Phosphatidic acid ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,030220 oncology & carcinogenesis ,Second messenger system ,Signal transduction ,Reactive Oxygen Species ,Mitochondrial ADP, ATP Translocases ,Intracellular - Abstract
Lipid-protein interactions play essential roles in many biological phenomena. Lysophospholipid mediators, such as cyclic phosphatidic acid (cPA), have been recognized as secondary messengers, yet few cellular targets for cPA have been identified to date. Furthermore, the molecular mechanism that activates these downstream signaling events remains unknown. In this study, using metabolically stabilized cPA carba-derivative (2ccPA)-immobilized magnetic beads, we identified adenine nucleotide translocase 2 (ANT2) as a 2ccPA-interacting protein in microglial cells. 2ccPA was tested for its ability to inhibit apoptosis caused by phenylarsine oxide in microglial cells. This damage was significantly improved upon 2ccPA treatment, along with cell proliferation, apoptosis, reactive oxygen species production, and intracellular ATP levels. This is the first report to suggest the direct binding of 2ccPA to ANT2 in microglial cells and provides evidence for a new benefit of 2ccPA in protecting microglial cells from apoptotic death induced by the ANT2-mediated signaling pathway.
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- 2020
6. Qualitative and quantitative comparison of cyclic phosphatidic acid and its related lipid species in rat serum using hydrophilic interaction liquid chromatography with tandem-mass spectrometry
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Seiya Tanaka, Mari Gotoh, Hiromu Murofushi, Keiko Fukasawa, Shingo Nakajima, and Kimiko Murakami-Murofushi
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Male ,0301 basic medicine ,Phosphatidic Acids ,Tandem mass spectrometry ,01 natural sciences ,Biochemistry ,Citric Acid ,Analytical Chemistry ,Palmitic acid ,03 medical and health sciences ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Phosphatidylcholine ,Animals ,heterocyclic compounds ,Rats, Wistar ,chemistry.chemical_classification ,Chromatography ,Hydrophilic interaction chromatography ,010401 analytical chemistry ,Organic Chemistry ,Reproducibility of Results ,Fatty acid ,General Medicine ,Phosphatidic acid ,Reference Standards ,0104 chemical sciences ,030104 developmental biology ,chemistry ,Phosphatidylcholines ,cardiovascular system ,lipids (amino acids, peptides, and proteins) ,Hydrochloric Acid ,Stearic acid ,Lysophospholipids ,Citric acid ,Hydrophobic and Hydrophilic Interactions ,Chromatography, Liquid - Abstract
Cyclic phosphatidic acid (cPA) is a simple lipid containing a fatty acid attached at the sn-1 position and a cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. The pharmacological effects of cPA have been demonstrated in several diseases such as cancer and neuropathic pain; however, the composition of the molecular species of cPA in relative to other lipid species in biological samples is still unclear. Recently, hydrophilic interaction liquid chromatography (HILIC) has demonstrated the ability to perform lipidomic analyses of biological samples. In the present study, we developed the quantitative measurement of cPA and its related lipid species, such as lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC), in rat serum using HILIC equipped with tandem-mass spectrometry (MS/MS). The lipid analysis using HILIC-MS/MS system demonstrated high linearity and reproducibility. The modified Bligh and Dyer method using citric acid was showed high efficiency on the extraction of cPA and LPA without contamination of artificial products. In rat serum, cPA and LPC contained more saturated fatty acids such as palmitic acid and stearic acid than unsaturated fatty acids, whereas LPA and phosphatidylcholine more contained unsaturated fatty acids than saturated fatty acids. The analytical methods for measuring cPA and its related lipid species in the present study will aid the analysis of their metabolism.
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- 2018
7. 2-carba cyclic phosphatidic acid suppresses inflammation via regulation of microglial polarisation in the stab-wounded mouse cerebral cortex
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Hiroko Ikeshima-Kataoka, Kimiko Murakami-Murofushi, Kei Hashimoto, Mari Gotoh, Ayana Hamano, Mari Nakashima, and Yasunori Miyamoto
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0301 basic medicine ,Traumatic brain injury ,Interleukin-1beta ,Phosphatidic Acids ,lcsh:Medicine ,Inflammation ,Pharmacology ,Real-Time Polymerase Chain Reaction ,Article ,Proinflammatory cytokine ,Transforming Growth Factor beta1 ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Brain Injuries, Traumatic ,medicine ,Animals ,RNA, Messenger ,Stab wound ,lcsh:Science ,Multidisciplinary ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,business.industry ,lcsh:R ,Phosphatidic acid ,medicine.disease ,Immunohistochemistry ,Extravasation ,030104 developmental biology ,Real-time polymerase chain reaction ,chemistry ,Blood-Brain Barrier ,Female ,lcsh:Q ,Microglia ,medicine.symptom ,business - Abstract
Traumatic brain injury (TBI) is caused by physical damage to the brain and it induces blood-brain barrier (BBB) breakdown and inflammation. To diminish the sequelae of TBI, it is important to decrease haemorrhage and alleviate inflammation. In this study, we aimed to determine the effects of 2-carba-cyclic phosphatidic acid (2ccPA) on the repair mechanisms after a stab wound injury as a murine TBI model. The administration of 2ccPA suppressed serum immunoglobulin extravasation after the injury. To elucidate the effects of 2ccPA on inflammation resulting from TBI, we analysed the mRNA expression of inflammatory cytokines. We found that 2ccPA prevents a TBI-induced increase in the mRNA expression of Il-1β, Il-6, Tnf-α and Tgf-β1. In addition, 2ccPA reduces the elevation of Iba1 levels. These data suggest that 2ccPA attenuates the inflammation after a stab wound injury via the modulation of pro-inflammatory cytokines release from microglial cells. Therefore, we focused on the function of 2ccPA in microglial polarisation towards M1 or M2 phenotypes. The administration of 2ccPA decreased the number of M1 and increased the number of M2 type microglial cells, indicating that 2ccPA modulates the microglial polarisation and shifts them towards M2 phenotype. These data suggest that 2ccPA treatment suppresses the extent of BBB breakdown and inflammation after TBI.
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- 2018
8. LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model
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Keisuke Yoshikawa, Tamotsu Tsukahara, Kimiko Murakami-Murofushi, Hayakazu Sumida, Masatake Araki, Satoshi Ishii, Ken Ichi Yamamura, Noriyuki Akahoshi, Ryoko Tsukahara, Keisuke Yanagida, Hiroyuki Neyama, Mari Gotoh, Shinji Yamamoto, Hiroshi Ueda, and Kimi Araki
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Male ,0301 basic medicine ,Multiple Sclerosis ,Receptor expression ,Gene Expression ,Mice, Inbred Strains ,Corpus callosum ,LPA1 Receptor ,Corpus Callosum ,Cuprizone ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Peripheral nerve ,Lysophosphatidic acid ,medicine ,Animals ,Receptors, Lysophosphatidic Acid ,Pharmacology ,business.industry ,Multiple sclerosis ,lcsh:RM1-950 ,medicine.disease ,Disease Models, Animal ,lcsh:Therapeutics. Pharmacology ,030104 developmental biology ,chemistry ,Hyperalgesia ,Neuropathic pain ,Neuralgia ,Molecular Medicine ,Female ,Lysophospholipids ,medicine.symptom ,business ,Neuroscience ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain. Keywords: LPA5, Neuropathic pain, Multiple sclerosis
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- 2018
9. 2- O -Carba-oleoyl cyclic phosphatidic acid induces glial proliferation through the activation of lysophosphatidic acid receptor
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Hiromu Murofushi, Shingo Nakajima, Kimiko Murakami-Murofushi, Keiko Fukasawa, and Mari Gotoh
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0301 basic medicine ,MAPK/ERK pathway ,Population ,Phosphatidic Acids ,03 medical and health sciences ,chemistry.chemical_compound ,Lysophosphatidic acid ,Animals ,RNA, Messenger ,Receptors, Lysophosphatidic Acid ,education ,Receptor ,Molecular Biology ,Cells, Cultured ,Cell Proliferation ,Oligodendrocyte Precursor Cells ,Mice, Inbred ICR ,education.field_of_study ,DNA synthesis ,Cell growth ,General Neuroscience ,Lipid signaling ,Phosphatidic acid ,Mitochondria ,Cell biology ,030104 developmental biology ,chemistry ,Astrocytes ,lipids (amino acids, peptides, and proteins) ,Neurology (clinical) ,Lysophospholipids ,Signal Transduction ,Developmental Biology - Abstract
Lysophosphatidic acid (LPA) and cyclic phosphatidic acid (cPA) are one of the lipid mediators regulating cell proliferation and differentiation through the activation of LPA receptors. An LPA receptor-mediated signal is important for the development of the central nervous system, while it has been demonstrated that LPA caused microglial activation and astroglial dysfunction. Previously, we have reported that cPA and carba analog of cPA, 2-O-carba-cPA (2ccPA), protected neural damage caused by transient ischemia. However, little is known about the target cell of cPA/2ccPA in the central nervous systems. Here, we examined the effect of 2ccPA on glial proliferation and differentiation using the primary astrocytes and oligodendrocyte precursor cells (OPCs) cultures. 2ccPA increased the DNA synthesis of astrocytes and OPCs, but it did not reduce the formazan production in the mitochondria. Further, 2ccPA increased the cell number and cell survival against oxidative stress. The inhibition of LPA receptors by ki16425 abolished 2ccPA-induced DNA synthesis. Extracellular signal-regulated kinase (ERK) was activated by 2ccPA, which contributed to the astroglial DNA synthesis. These results suggest that 2ccPA is a beneficial regulator of glial population through the activation of LPA receptor without reduction of mitochondrial activity.
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- 2018
10. Age-related changes in cyclic phosphatidic acid-induced hyaluronic acid synthesis in human fibroblasts
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Mari Gotoh, Kimiko Murakami-Murofushi, Noriaki Tajima, Katsura Sano, Yoshibumi Shimizu, and Kyoko Dodo
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Adult ,Male ,0301 basic medicine ,Aging ,Cancer Research ,medicine.medical_specialty ,Gene Expression ,Phosphatidic Acids ,Human skin ,Extracellular matrix ,Heterocyclic Compounds, 1-Ring ,Young Adult ,03 medical and health sciences ,chemistry.chemical_compound ,Dermis ,Internal medicine ,Hyaluronic acid ,medicine ,Humans ,RNA, Messenger ,Hyaluronic Acid ,Child ,Cells, Cultured ,Aged ,HAS1 ,biology ,Infant ,Cell Biology ,Phosphatidic acid ,Fibroblasts ,Stimulation, Chemical ,Hyaluronan synthase ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Biochemistry ,chemistry ,biology.protein ,Versican ,Female ,Hyaluronan Synthases - Abstract
Hyaluronic acid is a major component of the extracellular matrix, which is important for skin hydration. As aging brings skin dehydration, we aimed to clarify the mRNA expression of hyaluronic acid-related proteins in human skin fibroblasts from donors of various ages (range 0.7-69 years). Previously, we reported that cyclic phosphatidic acid (cPA), a unique phospholipid mediator, stimulated the expression of HAS2 and increased hyaluronic acid synthesis in human skin fibroblasts (donor age: 3 days). In this study, we measured the mRNA expression of hyaluronic acid-related proteins: hyaluronan synthase (HAS) 1-3, hyaluronidase-1, -2, and hyaluronic acid-binding protein (versican). In addition, we tested whether cPA could increase hyaluronic acid synthesis in skin fibroblasts derived from donors of various ages. The expression of HAS1, 3, hyaluronidase-1, and -2 did not change with aging. However, the mRNA expression of versican decreased with aging. Although it is thought that the amount of hyaluronic acid in the dermis decreases with aging, the mRNA expression of HAS2 was increased. But the amount of hyaluronic acid secreted by fibroblasts did not increase with aging. This suggests that the activity and/or protein expression of HAS2 decrease with aging. Furthermore, we observed that cPA caused the increase of hyaluronic acid synthesis at any age, and this effect was increased with aging. These results suggest that aging made the fibroblasts more sensitive to cPA treatment. Therefore, cPA represents a suitable candidate for the health maintenance and improvement of the skin by increasing the level of hyaluronic acid in the dermis.
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- 2017
11. Evaluation of the pharmacokinetics of 2-carba-cyclic phosphatidic acid by liquid chromatography-triple quadrupole mass spectrometry
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Yuki Shibaike, Mari Gotoh, Ryoko Tsukahara, Kensuke Iwasa, Shinji Yamamoto, Keisuke Yoshikawa, Keiko Fukasawa, Yoshibumi Shimizu, Kimiko Murakami-Murofushi, and Masaki Ishikawa
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0301 basic medicine ,Male ,Physiology ,Phosphatidic Acids ,030204 cardiovascular system & hematology ,Pharmacology ,Mass spectrometry ,Biochemistry ,Neuroprotection ,Mass Spectrometry ,Rats, Sprague-Dawley ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Pharmacokinetics ,Oral administration ,Lysophosphatidic acid ,medicine ,Animals ,Tissue Distribution ,Stomach ,Capsule ,Cell Biology ,Phosphatidic acid ,Rats ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Chromatography, Liquid - Abstract
Cyclic phosphatidic acid (cPA) is a lysophospholipid mediator that suppresses cancer metastasis and osteoarthritis. It also has neuroprotective roles in diseases such as multiple sclerosis and delayed neuronal death following transient ischemia. In order to take advantage of the properties of cPA for the development of new therapeutic strategies, we have synthesized several cPA derivatives and discovered 2-carba-cPA (2ccPA) as a promising candidate. To develop 2ccPA as a therapeutic agent, we investigated the pharmacokinetic profile of 2ccPA by liquid chromatography-triple quadrupole mass spectrometry in this study. When 2ccPA was administered intraperitoneally to mice at a dose of 1.6 mg/kg, the half-life of 2ccPA in plasma was 16 min. The 2ccPA, dosed intraperitoneally to mice at 16 mg/kg, distributed to each organ including brain at 20 min after dosing. It was found that 2ccPA was stable in neutral or alkaline conditions (e.g., intestine) but unstable in acidic conditions (e.g., stomach). When 2ccPA was orally administrated to rats as a gastro-resistant form using an enterosoluble capsule, plasma 2ccPA levels peaked at 2 h, slowly declined thereafter and persistently detected even at 10 h after administration. Here, we present the findings on the effect of the continuous release of 2ccPA from the capsule to reduce the lysophospholipase D activity and also decrease plasma levels of lysophosphatidic acid in rat. These findings will be useful in further studies for evaluating the application of 2ccPA in several disorders.
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- 2019
12. 2-Carba cyclic phosphatidic acid inhibits lipopolysaccharide-induced prostaglandin E2 production in a human macrophage cell line
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Keisuke Yoshikawa, Shingo Nakajima, Mari Gotoh, Kimiko Murakami-Murofushi, Yuki Shibaike, Chinatsu Ogawa, and Tetsuyuki Kobayashi
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0301 basic medicine ,Lipopolysaccharide ,Prostaglandin E2 receptor ,medicine.medical_treatment ,Prostaglandin E2 ,Biophysics ,2ccPA ,Biochemistry ,lcsh:Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,lcsh:QD415-436 ,THP-1 monocytes ,lcsh:QH301-705.5 ,Neuroinflammation ,Experimental autoimmune encephalomyelitis ,Phosphatidic acid ,medicine.disease ,Molecular biology ,030104 developmental biology ,Cytokine ,chemistry ,lcsh:Biology (General) ,030220 oncology & carcinogenesis ,Phorbol ,lipids (amino acids, peptides, and proteins) ,Anti-inflammatory ,medicine.drug ,Research Article - Abstract
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that contains a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Using mouse models for multiple sclerosis (cuprizone-induced demyelination and experimental autoimmune encephalomyelitis) and traumatic brain injury, we revealed that cPA and its metabolically stabilized cPA derivative, 2-carba-cPA (2ccPA), have potential to protect against neuroinflammation. In this study, we investigated whether 2ccPA has anti-inflammatory effect on peripheral immune function or not using inflammation-induced macrophages-like cell line, THP-1 monocytes differentiated by phorbol 12-myristate 13-acetate (PMA). Lipopolysaccharide (LPS)-stimulated THP-1 cells were found to have higher expression of the mRNAs of several inflammation-related cytokines and of the enzyme cyclooxygenase-2 (Cox-2); however, when THP-1 cells were stimulated by LPS in the presence of 2ccPA, the increase in the expression of pro-inflammatory cytokine and Cox-2 mRNA was attenuated. 2ccPA treatment also decreased the amount of prostaglandin E2 (PGE2) produced by LPS-stimulated THP-1 cells and decreased expression of the mRNA of prostaglandin E receptor 2 (EP2, PTGER2), a PGE2 receptor that mediates inflammation. These results indicate that 2ccPA has anti-inflammatory properties., Highlights • 2-Carba cyclic phosphatidic acid inhibits prostaglandin E2 production. • 2-Carba cyclic phosphatidic acid has anti-inflammatory effect. • 2-Carba cyclic phosphatidic acid has effect on peripheral immune function.
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- 2019
13. Oleic acid is a potent inducer for lipid droplet accumulation through its esterification to glycerol by diacylglycerol acyltransferase in primary cortical astrocytes
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Hiroshi Kunugi, Shingo Nakajima, Mari Gotoh, Keiko Fukasawa, and Kimiko Murakami-Murofushi
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0301 basic medicine ,Glycerol ,MAP Kinase Signaling System ,Primary Cell Culture ,Phospholipid ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Lipid droplet ,Lipidomics ,Extracellular ,Animals ,Diacylglycerol O-Acyltransferase ,Molecular Biology ,Cerebral Cortex ,Mice, Inbred ICR ,Esterification ,General Neuroscience ,Lipid metabolism ,Lipid Droplets ,Lipid Metabolism ,Oleic acid ,030104 developmental biology ,chemistry ,Biochemistry ,Lipotoxicity ,Astrocytes ,Ketone bodies ,lipids (amino acids, peptides, and proteins) ,Neurology (clinical) ,Proto-Oncogene Proteins c-akt ,030217 neurology & neurosurgery ,Developmental Biology ,Oleic Acid - Abstract
Astrocytes exhibit an important role in neural lipid metabolism for the regulation of energy balance to supply fatty acids (FAs) and ketone bodies to other neural cells. Lipid droplets (LDs) consisting of neutral- and phospho-lipids increase in the brains of patients with neurodegenerative diseases, such as Alzheimer’s disease and multiple sclerosis. However, the role of LDs and its lipid source remains largely unexplored. Here, we found that oleic acid (OA) was a potent inducer of astrocytic LD accumulation among various FAs. Lipidomic analysis using liquid chromatography equipped with tandem mass spectrometry revealed that the cellular triacylglycerol and phospholipid compositions in astrocytes during LD accumulation reflected the condition of extracellular FAs. Furthermore, the inhibition of diacylglycerol acyltransferase blocked OA-induced LD accumulation and caused lipotoxicity-induced cell death in astrocytes. The present study demonstrated that the formation of LDs, caused due to the increased extracellular OA, facilitated survival against lipotoxic condition.
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- 2019
14. Correction to: Protective and therapeutic role of 2-carbacyclic phosphatidic acid in demyelinating disease
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Kei Maruyama, Kimiko Murakami-Murofushi, Masaki Ishikawa, Shinji Yamamoto, Mari Gotoh, Kota Yamashina, Keisuke Yoshikawa, Kensuke Iwasa, and Sosuke Yagishita
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0301 basic medicine ,business.industry ,General Neuroscience ,Immunology ,Phosphatidic acid ,medicine.disease ,lcsh:RC346-429 ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,Neurology ,chemistry ,Demyelinating disease ,Medicine ,business ,Neuroscience ,lcsh:Neurology. Diseases of the nervous system ,030217 neurology & neurosurgery - Abstract
After publication of the article [1], it was brought to our attention that an acknowledgement was missing from the original version.
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- 2018
15. Corrigendum to 'Quantitative determination of cyclic phosphatidic acid and its carba analog in mouse organs and plasma using LC–MS/MS' [J. Chromatogr. B 1076 (2018) 15–21]
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Kensuke Iwasa, Shinji Yamamoto, Keisuke Yoshikawa, Yoshibumi Shimizu, Kimiko Murakami-Murofushi, Mari Gotoh, Masaki Ishikawa, and Keiko Fukasawa
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chemistry.chemical_compound ,Chromatography ,Chemistry ,Clinical Biochemistry ,Lc ms ms ,Cell Biology ,General Medicine ,Phosphatidic acid ,Biochemistry ,Quantitative determination ,Analytical Chemistry - Published
- 2018
16. Pharmacological evaluation of a novel cyclic phosphatidic acid derivative 3-S-cyclic phosphatidic acid (3-S-cPA)
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Emi Nozaki, Ryo Tanaka, Susumu Kobayashi, Atsuo Nakazaki, Harumi Hotta, Kimiko Murakami-Murofushi, Mari Gotoh, Masaru Kato, and Takahiro Suzuki
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Male ,Programmed cell death ,Stereochemistry ,Clinical Biochemistry ,Phospholipid ,Phosphatidic Acids ,Pharmaceutical Science ,Ring (chemistry) ,Biochemistry ,Fatty Acids, Monounsaturated ,chemistry.chemical_compound ,Cell Movement ,Drug Discovery ,Animals ,Humans ,heterocyclic compounds ,Rats, Wistar ,Molecular Biology ,Neurons ,Cell Death ,Molecular Structure ,Phosphoric Diester Hydrolases ,Organic Chemistry ,Stereoisomerism ,Phosphatidic acid ,Phosphate ,Rats ,chemistry ,cardiovascular system ,Molecular Medicine ,Enantiomer ,Autotaxin ,Chirality (chemistry) - Abstract
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator possessing cyclic phosphate ring, which is necessary for its specific biological activities. To stabilize cyclic phosphate ring of cPA, we synthesized a series of cPA derivatives. We have shown that racemic 3-S-cPA, with a phosphate oxygen atom replaced with a sulfur atom at the sn-3, was a more effective autotaxin (ATX) inhibitor than cPA. In this study, we showed that racemic 3-S-cPA also had potent biological activities such as inhibition of cancer cell migration, suppression of the nociceptive reflex, and attenuation of ischemia-induced delayed neuronal cell death in the hippocampal CA1. Moreover, we synthesized both enantiomers of palmitoleoyl derivative of 3-S-cPA, and found that the chirality of 3-S-cPA is not involved in ATX inhibition. Based on these findings, racemic 3-S-cPA is suggested as an effective therapeutic compound like cPA.
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- 2012
17. A Possible Mechanism of Cholesteryl Glucoside Formation Involved in Heat Shock Response in the Animal Cell Membrane
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Hisako Akiyama, Kimiko Murakami-Murofushi, Susumu Kobayashi, Tsutomu Hamada, Yasuko Nagatsuka, and Yoshio Hirabayashi
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Cell Biology ,Plant Science ,Lipid signaling ,Biology ,Cell biology ,Hsp70 ,Heat shock factor ,Cell membrane ,Glycolipid ,medicine.anatomical_structure ,Biochemistry ,Genetics ,medicine ,lipids (amino acids, peptides, and proteins) ,Animal Science and Zoology ,Heat shock ,HSF1 ,Lipid raft - Abstract
We previously reported that cholesteryl glucoside (CG), a member of membrane glycolipid, is rapidly induced by exposure to some forms of stress in animal tissues and human cultured cells. As CG is induced by heat shock before the activation of heat shock transcription factor 1 (HSF1) and the production of heat shock protein 70 (HSP70), and CG added exogenously induces HSF1 activation and HSP70 production in animal tissue and human fibroblasts, it is suggested that CG functions as a crucial lipid mediator in cellular responses against heat stress.In this report, we showed the localization of CG synthetase, sterol glucosyltransferase, at lipid raft in the human cell membrane. Because the lipid raft is considered to be a scaffold of heat shock response leading to HSP induction, we propose that CG formation by sterol glucosyltransferase in lipid raft might act as a potential factor in the thermal sensing reaction. Additionally, using the artificial liposomes modeling on the states of membranes before and after CG production, we clarified that the transfer of the glucose moiety from glucose donor, glucosylceramide, to cholesterol changed membrane physical properties and formed thermostable solid-ordered domains. We suggest that the alteration of membrane physical state caused by heat stress might be linked to activate sterol glucosyltransferase to form CG in the animal.
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- 2011
18. Cyclic phosphatidic acid decreases proliferation and survival of colon cancer cells by inhibiting peroxisome proliferator-activated receptor γ
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Kimiko Murakami-Murofushi, Tamotsu Tsukahara, Shuwa Hanazawa, Yoshiki Iwamoto, and Tetsuyuki Kobayashi
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medicine.medical_specialty ,Cell type ,Cell Survival ,Physiology ,Phosphatidic Acids ,Peroxisome proliferator-activated receptor ,Biology ,Biochemistry ,Heterocyclic Compounds, 1-Ring ,chemistry.chemical_compound ,Cell Line, Tumor ,Internal medicine ,Lysophosphatidic acid ,medicine ,Humans ,heterocyclic compounds ,Receptor ,Cell Proliferation ,Pharmacology ,chemistry.chemical_classification ,Cell growth ,DNA ,Cell Biology ,Phosphatidic acid ,Peroxisome ,PPAR gamma ,Endocrinology ,chemistry ,Nuclear receptor ,Colonic Neoplasms ,cardiovascular system ,Cancer research ,Lysophospholipids ,HT29 Cells - Abstract
Cyclic phosphatidic acid (cPA), a structural analog of lysophosphatidic acid (LPA), is one of the simplest phospholipids found in every cell type. cPA is a specific, high-affinity antagonist of peroxisome proliferator-activated receptor gamma (PPARγ); however, the molecular mechanism by which cPA inhibits cellular proliferation remains to be clarified. In this study, we found that inhibition of PPARγ prevents proliferation of human colon cancer HT-29 cells. cPA suppressed cell growth, and this effect was reversed by the addition of a PPARγ agonist. These results indicate that the physiological effects of cPA are partly due to PPARγ inhibition. Our results identify PPARγ as a molecular mediator of cPA activity in HT-29 cells, and suggest that cPA and the PPARγ pathway might be therapeutic targets in the treatment of colon cancer.
- Published
- 2010
19. Lysophosphatidic acid-activated Cl- current activity in human systemic sclerosis skin fibroblasts
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Arnold E. Postlethwaite, Gabor Tigyi, Zhaohong Yin, Kimiko Murakami-Murofushi, Mari Gotoh, Laura D Carbone, Alyssa L. Bolen, and Mitchell A. Watsky
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Adult ,Male ,medicine.medical_specialty ,Pathology ,Systemic scleroderma ,chemistry.chemical_compound ,Chlorides ,Basic Science ,Rheumatology ,Fibrosis ,Internal medicine ,Lysophosphatidic acid ,medicine ,Humans ,Pharmacology (medical) ,Myofibroblasts ,skin and connective tissue diseases ,Fibroblast ,Cells, Cultured ,Skin ,Autoimmune disease ,Analysis of Variance ,Scleroderma, Systemic ,integumentary system ,business.industry ,Muscle, Smooth ,Fibroblasts ,Middle Aged ,medicine.disease ,Connective tissue disease ,Actins ,Blot ,medicine.anatomical_structure ,Endocrinology ,chemistry ,Case-Control Studies ,Female ,Lysophospholipids ,business ,Myofibroblast - Abstract
Objectives. SSc (scleroderma) is an often fatal disease characterized by widespread tissue fibrosis. Fibroblasts play a key role in SSc-associated fibrosis. This study was designed to determine: (i) whether fibroblasts isolated from skin of patients with SSc have increased lysophosphatidic acid-activated Cl - current (ICI LPA ) activity vs healthy controls; (ii) whether myofibroblast differentiation is involved in SSc skin fibrosis; and (iii) whether SSc fibroblasts have different proliferation rates vs controls. Methods. Skin biopsies were taken from involved and uninvolved skin of SSc patients and controls. Whole-cell perforated patch-clamping was used to measure ICI LPA activity in fibroblasts isolated and cultured from these biopsies. Western blotting was used to measure α-smooth muscle actin (α-SMA). Proliferation was measured using a colorimetric assay. Results. Fibroblasts cultured from SSc skin show significantly increased ICI LPA activity following LPA exposure compared with control skin fibroblasts. α-SMA protein was significantly increased in cultured SSc skin fibroblasts vs controls. No significant differences in proliferation rates were found. Conclusions. Elevated ICI LPA activity is a hallmark of SSc skin fibroblasts. Blocking ICI LPA activation may be a new therapeutic approach for treating SSc-associated fibrosis.
- Published
- 2010
20. Phospholipase D2-Dependent Inhibition of the Nuclear Hormone Receptor PPARγ by Cyclic Phosphatidic Acid
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Tetsuyuki Kobayashi, Michael A. Frohman, Yuko Fujiwara, Chunxiang Zhang, Alyssa L. Bolen, Ryoko Tsukahara, Fabio Re, Gabor Tigyi, Abby L. Parrill, Ayako Uchiyama, Junming Yue, Kimiko Murakami-Murofushi, Tamotsu Tsukahara, Yunhui Cheng, Guangwei Du, Daniel L. Baker, Huazhang Guo, and Louisa Balazs
- Subjects
Transcription, Genetic ,Peroxisome proliferator-activated receptor ,Phosphatidic Acids ,Phospholipase ,Biology ,Article ,chemistry.chemical_compound ,Mice ,3T3-L1 Cells ,Adipocytes ,Phospholipase D ,Animals ,heterocyclic compounds ,Nuclear Receptor Co-Repressor 2 ,Molecular Biology ,Nuclear receptor co-repressor 2 ,chemistry.chemical_classification ,PLD2 ,Macrophages ,Cell Differentiation ,Phosphatidic acid ,Cell Biology ,Rats ,PPAR gamma ,chemistry ,Biochemistry ,Nuclear receptor ,Second messenger system ,cardiovascular system - Abstract
Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.
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- 2010
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21. Transcellular Invasion of MM1 Rat Ascites Hepatoma Cells Requires Matrix Metalloproteinases Derived from Host Mesothelium
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Yukari Arai, Kimiko Murakami-Murofushi, Gabor Tigyi, Tetsuyuki Kobayashi, and Ayako Uchiyama
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Proteases ,Tumor microenvironment ,Cell Biology ,Plant Science ,Matrix metalloproteinase ,Biology ,In vitro ,Cell biology ,Extracellular matrix ,Mesothelium ,medicine.anatomical_structure ,Genetics ,medicine ,Animal Science and Zoology ,Transcellular ,Mesothelial Cell - Abstract
Degradation of the extracellular matrix by proteases secreted by invading tumor cells is thought to be essential for metastasis. Using an in vitro transcellular migration assay model, we examined the requirement of matrix metalloproteinases (MMP) in the invasion of MM1 rat hepatoma cells through normal mesothelial cell monolayers. Here we show that MM1 cells transmigrate using MMPs not expressed in the tumor cells but secreted by host mesothelial cells. Additionally, the amount of MMP secreted by mesothelial cells was increased by co-culture with hepatoma cells. Our results point to the role of normal host cells in the tumor microenvironment as a source of invasion factors necessary for the metastatic process.
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- 2010
22. Inhibition of Human Tumor Cell Proliferation by the Telomerase Inhibitor TELIN
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Yasutaka Kakiuchi, Hiromu Murofushi, Mayuka Nakatake, Hidetoshi Arima, Yayoi Okamoto, Hirofumi Kai, Kimiko Murakami-Murofushi, and Mika Oyama
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Telomerase ,Cell growth ,Cell ,Cell Biology ,Plant Science ,Biology ,Molecular biology ,Telomere ,Human tumor ,medicine.anatomical_structure ,Apoptosis ,Delivery efficiency ,Genetics ,medicine ,Animal Science and Zoology ,Telomerase reverse transcriptase - Abstract
TELIN, a newly developed catalytic blocker of telomerase, was assessed for its antiproliferative activity against human tumor cells in in vitro cell culture assay. TELIN suppressed cell proliferations of several blood tumor cells with telomere shortening and the appearance of apoptotic small cells, but did not affect hTERT mRNA expression, suggesting that TELIN catalytically inhibited telomerase activity within the cells. TELIN also suppressed cell proliferation of adherent tumor cells, whereas it did not affect the growth of normal fibroblasts. Delivery efficiency of TELIN was significantly different between several delivery carriers, and β-cyclodextrin was found to be a suitable carrier.
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- 2010
23. Cdc42/N-WASP and Rac1/WAVE2 Required for LPA-induced Migration of Rat Ascites Hepatoma Cells
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Kimiko Murakami-Murofushi, Gabor Tigyi, and Ayako Uchiyama
- Subjects
Shape change ,RAC1 ,Cell migration ,macromolecular substances ,Cell Biology ,Plant Science ,CDC42 ,Biology ,Cell biology ,chemistry.chemical_compound ,chemistry ,Ascites ,Lysophosphatidic acid ,Genetics ,medicine ,lipids (amino acids, peptides, and proteins) ,Animal Science and Zoology ,biological phenomena, cell phenomena, and immunity ,medicine.symptom ,Transcellular ,Mesothelial Cell - Abstract
Lysophosphatidic acid (LPA) has been shown to stimulate transcellular migration of MM1 rat ascites hepatoma cells through transient activation of the Rho/ROCK pathway. Here we report that LPA also induces transient inactivation of Cdc42 and Rac1, both of which are required for transmigration of MM1 cells. Cdc42-induced shape change is a required step for LPA-induced MM1 cell migration. We provide evidence that MM1 cells transmigrate the mesothelial cell monolayer through activation of not only previously reported Rho/ROCK pathway but also Rac/WAVE2 and Cdc42/N-WASP pathways.
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- 2010
24. MOV10 as a novel telomerase-associated protein
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Mika Ohyama, Toshinori Ide, Mariko Nakano, Akihiro Iwamatsu, Kazuo Kobayashi, Yukiko Shimada, Yasutaka Kakiuchi, Ei Yamada, Naoko Yano, Narie Sasaki-Higashiyama, Kazuhiko Kaji, Kimiko Murakami-Murofushi, Fumi Ikeda, Hiromu Murofushi, Sohsuke Ichikawa, Kiyoto Nishiyama, and Yasuko Ogiwara
- Subjects
Male ,Telomerase ,Swine ,Biophysics ,Biology ,Biochemistry ,Telomerase RNA component ,chemistry.chemical_compound ,Testis ,Animals ,Humans ,Telomerase reverse transcriptase ,Cloning, Molecular ,Molecular Biology ,Gene Library ,Telomere-binding protein ,Messenger RNA ,cDNA library ,DNA ,Cell Biology ,Telomere ,Molecular biology ,chemistry ,RNA Helicases ,HeLa Cells - Abstract
A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.
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- 2009
25. ThePlasmodiumHU homolog, which binds the plastid DNA sequence-independent manner, is essential for the parasite's survival
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Makoto Hirai, Masayuki Hata, Shigeharu Sato, Ryoko Yui, Syoko Namiki, Kie Itoh, Narie Sasaki, Teppei Morita, Kiyoshi Kita, Katsura Maeda, Kimiko Murakami-Murofushi, and Hiroyuki Matsuoka
- Subjects
Plastid ,Plasmodium berghei ,Plasmodium falciparum ,HU Protein ,Protozoan Proteins ,Biophysics ,Biochemistry ,Plasmodium ,Genome ,DNA sequencing ,Gene Knockout Techniques ,HU ,Structural Biology ,parasitic diseases ,Genetics ,Animals ,Plastids ,Molecular Biology ,Gene ,DNA Primers ,Base Sequence ,biology ,Genetic Complementation Test ,Cell Biology ,DNA, Protozoan ,biology.organism_classification ,Molecular biology ,DNA binding protein ,Knock-out - Abstract
The nuclear genome of the human malaria parasite Plasmodium falciparum encodes a homolog of the bacterial HU protein (PfHU). In this study, we characterised PfHU’s physiological function. PfHU, which is targeted exclusively to the parasite’s plastid, bound its natural target – the plastid DNA – sequence-independently and complemented lack of HU in Escherichia coli. The HU gene could not be knocked-out from the genome of Plasmodium berghei, implying that HU is important for the parasite’s survival. As the human cell lacks the HU homolog, PfHU is a potential target for drugs to control malaria.
- Published
- 2009
26. Visualization of Mitochondrial and Apicoplast Nucleoids in the Human Malaria Parasite Plasmodium falciparum by SYBR Green I and PicoGreen Staining
- Author
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Kie Ito, Masayuki Hata, Kiyoshi Kita, Takashi Ueda, Narie Sasaki, Akihiko Nakano, Shigeharu Sato, Ryoko Yui, Katsura Maeda-Sano, and Kimiko Murakami-Murofushi
- Subjects
Apicoplast ,animal structures ,biology ,fungi ,Plasmodium falciparum ,Cell Biology ,Plant Science ,biology.organism_classification ,Genome ,Molecular biology ,Plasmodium ,Cell biology ,chemistry.chemical_compound ,chemistry ,embryonic structures ,parasitic diseases ,Genetics ,SYBR Green I ,bacteria ,Parasite hosting ,Nucleoid ,Animal Science and Zoology ,Plastid - Abstract
Organellar DNA in mitochondria and plastids are organized with proteins into a compact structure known as the nucleoid. As the nucleoid is supposed to be the unit of inheritance for the organellar genome, it is important to understand its cytological behavior. Like plants, malaria parasites carry two organelles, the mitochondrion and the apicoplast–a non-photosynthetic plastid. However, probably because of the small size of the genome in each, visualizing the nucleoid in the Plasmodium organelles by regularly-used fluorescent dye such as DAPI, has been difficult. Here, we developed new, effective methods to visualize the organellar nucleoid in the human malaria parasite Plasmodium falciparum. With our methods using SYBR Green I or PicoGreen, nucleoids were observed in ring-stage parasites. Analyzing transfectant parasites carrying a DsRed-labelled organelle, we concluded that the parasite's mitochondrion has 1 nucleoid which is visualized with our methods. The parasite has a second nucleoid in the apicoplast, but higher concentration of the dye was required to visualize it. Our new methods would be useful for further cytological analysis of the nucleoids in the mitochondrion and the apicoplast of the malaria parasite.
- Published
- 2009
27. New Protein Pmn34 with an Exonuclease Motif Localizes in the Mitochondrial Nucleoid Periphery of Physarum polycephalum
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Masahiro M. Kanaoka, Haruko Kuroiwa, Akiko Izumi, Tetsuya Higashiyama, Narie Sasaki, Tsuneyoshi Kuroiwa, Shigeyuki Kawano, Kimiko Murakami-Murofushi, Katsura Maeda-Sano, Naoshi Dohmae, Ryoko Yui, Toshiyuki Mori, and Kie Itoh
- Subjects
Exonuclease ,Mitochondrial DNA ,animal structures ,DEDD ,Physarum polycephalum ,Plant Science ,Biology ,law.invention ,chemistry.chemical_compound ,law ,Genetics ,Mitochondrial nucleoid ,chemistry.chemical_classification ,fungi ,Cell Biology ,biology.organism_classification ,Molecular biology ,Cell biology ,Amino acid ,chemistry ,embryonic structures ,Recombinant DNA ,biology.protein ,bacteria ,Animal Science and Zoology ,DNA - Abstract
Mitochondrial DNA (mtDNA) is highly organized into a compact structure, the mitochondrial nucleoid (mt-nucleoid). To facilitate our understanding of the regulation of mtDNA genetic activity within the mt-nucleoid structure, we have identified a novel mt-nucleoid protein Pmn34 (Physarum polycephalum mitochondrial nucleoid protein 34), having a molecular weight of 34 kDa, from pure mt-nucleoids isolated from the true slime mold, Physarum polycephalum. The Pmn34 protein is composed of 326 amino acids with mitochondrial transit peptides and its primary sequence contains a conserved 3′ to 5′ exonuclease motif of the “DEDD” superfamily. DNA mobility shift assays demonstrated that recombinant Pmn34 binds weakly to both mtDNA and λDNA with no apparent sequence specificity. Furthermore, immunoblotting and immunostaining analyses revealed that Pmn34 localizes specifically in the peripheral region of mt-nucleoids. These results indicate that Pmn34 functions in the peripheral region of mt-nucleoids, suggesting that the mt-nucleoid is compartmentalized into functional domains.
- Published
- 2009
28. Pertuzumab, a novel HER dimerization inhibitor, inhibits the growth of human lung cancer cells mediated by the HER3 signaling pathway
- Author
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Nagahiro Saijo, Kimiko Murakami-Murofushi, Kazuko Sakai, Tomohide Tamura, Hideyuki Yokote, and Kazuto Nishio
- Subjects
MAPK/ERK pathway ,Cancer Research ,medicine.medical_specialty ,Lung Neoplasms ,Receptor, ErbB-3 ,Neuregulin-1 ,Biology ,Antibodies, Monoclonal, Humanized ,Ligands ,Epidermal growth factor ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,Internal medicine ,medicine ,Humans ,Phosphorylation ,skin and connective tissue diseases ,Receptor ,Protein kinase B ,Cell Proliferation ,Epidermal Growth Factor ,Cell growth ,Antibodies, Monoclonal ,General Medicine ,Growth Inhibitors ,ErbB Receptors ,Endocrinology ,Oncology ,Cancer research ,Pertuzumab ,Signal transduction ,Dimerization ,Cell Division ,Signal Transduction ,medicine.drug - Abstract
A humanized anti-HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2-expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti-tumor activity of pertuzumab against eight non-small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)-alpha. Pertuzumab significantly inhibited the HRG-alpha-stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG-alpha-stimulated phosphorylation of HER3, mitogen-activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)-stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG-alpha-stimulated HER2/HER3 heterodimer formation. HRG-alpha-stimulated HER3 phosphorylation was also observed in the PC-9 cells co-overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG-alpha nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG-alpha-dependent cell growth in lung cancer cells through inhibition of HRG-alpha-stimulated HER2/HER3 signaling.
- Published
- 2007
29. Cyclic phosphatidic acid inhibits the secretion of vascular endothelial growth factor from diabetic human coronary artery endothelial cells through peroxisome proliferator-activated receptor gamma
- Author
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Yoshikazu Matsuda, Ryoko Tsukahara, Kimiko Murakami-Murofushi, Tamotsu Tsukahara, and Hisao Haniu
- Subjects
Neointima ,Vascular Endothelial Growth Factor A ,medicine.medical_specialty ,Peroxisome proliferator-activated receptor gamma ,Peroxisome proliferator-activated receptor ,Phosphatidic Acids ,Biology ,Biochemistry ,chemistry.chemical_compound ,Heterocyclic Compounds, 1-Ring ,Endocrinology ,Cell Movement ,Internal medicine ,medicine ,Humans ,Cyclic phosphatidic acid ,Receptor ,Diabetic human coronary artery endothelial cells ,Molecular Biology ,Cells, Cultured ,Cell Proliferation ,chemistry.chemical_classification ,Endothelial Cells ,Phosphatidic acid ,Atherosclerosis ,VEGF ,Coronary Vessels ,Vascular endothelial growth factor ,PPAR gamma ,chemistry ,Diabetes Mellitus, Type 2 ,Peroxisome proliferator-activated receptor alpha ,Lipoprotein - Abstract
Atherosclerosis is a disease characterized by building up plaques formation and leads to a potentially serious condition in which arteries are clogged by fatty substances such as cholesterol. Increasing evidence suggests that atherosclerosis is accelerated in type 2 diabetes. Recent study reported that high level of alkyl glycerophosphate (AGP) was accumulated in atherosclerotic lesions. The presence of this phospholipid in mildly oxidized low-density lipoprotein (LDL) is likely to be involved in atherogenesis. It has been reported that the activation of peroxisome proliferator-activated receptor gamma plays a key role in developing atherosclerosis. Our previous result indicates that cyclic phosphatidic acid (cPA), one of bioactive lipids, potently suppresses neointima formation by inhibiting the activation of peroxisome proliferator-activated receptor gamma (PPARγ). However, the detailed mechanism is still unclear. In this study, to elucidate the mechanism of the cPA-PPARγ axis in the coronary artery endothelium, especially in patients with type 2 diabetes, we investigated the proliferation, migration, and secretion of VEGF in human coronary artery endothelial cells from diabetes patients (D-HCAECs). AGP induced cell growth and migration; however, cPA suppressed the AGP-elicited growth and migration in D-HCAECs. Moreover, AGP increased VEGF secretion from D-HCAECs, and this event was attenuated by cPA. Taken together, these results suggest that cPA suppresses VEGF-stimulated growth and migration in D-HCAECs. These findings could be important for regulatory roles of PPARγ and VEGF in the vascular processes associated with diabetes and atherosclerosis., Molecular and Cellular Endocrinology, 412, pp.320-329; 2015
- Published
- 2015
30. Cyclic Phosphatidic Acid Is Produced by Autotaxin in Blood
- Author
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Hiroyuki Arai, Tetsuyuki Kobayashi, Satomi Tsuda, Junken Aoki, Masayuki Tanaka, Shinichi Okudaira, Kimiko Murakami-Murofushi, Chie Shimamoto, and Keiko Moriya-Ito
- Subjects
Immunoprecipitation ,Sodium Chloride ,Ether ,Biochemistry ,chemistry.chemical_compound ,Western blot ,Multienzyme Complexes ,Lysophosphatidic acid ,medicine ,Animals ,Humans ,heterocyclic compounds ,Pyrophosphatases ,Molecular Biology ,chemistry.chemical_classification ,medicine.diagnostic_test ,Phosphoric Diester Hydrolases ,Antibodies, Monoclonal ,Lysophosphatidylcholines ,Cell Biology ,Phosphatidic acid ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Rats ,Blood ,Enzyme ,Lysophosphatidylcholine ,chemistry ,Metals ,Phosphodiesterase I ,cardiovascular system ,Cattle ,lipids (amino acids, peptides, and proteins) ,Lysophospholipids ,Autotaxin ,Fetal bovine serum - Abstract
Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid (LPA), was previously identified in human serum. Although cPA possesses distinct physiological activities not elicited by LPA, its biochemical origins have scarcely been studied. In the present study, we assayed cPA formation from lysophosphatidylcholine in fetal bovine serum and found significant activity of transphosphatidylation that generated cPA. The cPA-producing enzyme was purified from fetal bovine serum using five chromatographic steps yielding a 100-kDa protein with cPA biosynthetic activity. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of its tryptic peptides revealed that the enzyme shared identical fragments with human autotaxin, a serum lysophospholipase D that produces LPA. Western blot analysis demonstrated that the 100-kDa protein was specifically recognized by an anti-human autotaxin antibody. Moreover, recombinant rat autotaxin was found to generate cPA in addition to LPA. No significant cPA- or LPA-producing activity was detected in autotaxin-depleted serum from bovine or human prepared by immunoprecipitation with an anti-autotaxin monoclonal antibody. These results indicate that the generation of cPA and LPA in serum is mainly attributed to autotaxin.
- Published
- 2006
31. Carba Analogs of Cyclic Phosphatidic Acid Are Selective Inhibitors of Autotaxin and Cancer Cell Invasion and Metastasis
- Author
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Daniel L. Baker, Ryoko Tsukahara, Yuko Fujiwara, Robert Bittman, Hiromu Murofushi, Kimiko Murakami-Murofushi, Dominic Fan, Russell W. Bandle, Eunjin Koh, Gordon B. Mills, Gabor Tigyi, Susumu Kobayashi, Ayako Uchiyama, Mandi M. Murph, Kathryn R. Pigg, and Hoe-Sup Byun
- Subjects
Cell ,Phosphatidic Acids ,Antineoplastic Agents ,Biology ,Biochemistry ,Article ,Metastasis ,chemistry.chemical_compound ,Multienzyme Complexes ,In vivo ,Cell Line, Tumor ,Lysophosphatidic acid ,medicine ,Humans ,Neoplasm Invasiveness ,Neoplasm Metastasis ,Pyrophosphatases ,Receptor ,Melanoma ,Molecular Biology ,Phosphoric Diester Hydrolases ,Cell Biology ,Phosphatidic acid ,Lipid Metabolism ,medicine.disease ,Recombinant Proteins ,Spectrometry, Fluorescence ,medicine.anatomical_structure ,chemistry ,Phosphodiesterase I ,Culture Media, Conditioned ,Cancer cell ,Cancer research ,lipids (amino acids, peptides, and proteins) ,Lysophospholipids ,Autotaxin - Abstract
Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2, NPP2) is an autocrine motility factor initially characterized from A2058 melanoma cell conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.
- Published
- 2006
32. Regulation of levels of actin threonine phosphorylation during life cycle ofPhysarum polycephalum
- Author
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Narie Sasaki, Yuki Shirai, Kie Itoh, Tetsuyuki Kobayashi, Yoshiro Kishi, Kimiko Murakami-Murofushi, Akiko Izumi, and Masazumi Sameshima
- Subjects
Threonine ,Actin phosphorylation ,Blotting, Western ,Protozoan Proteins ,Arp2/3 complex ,Physarum polycephalum ,macromolecular substances ,Protein Serine-Threonine Kinases ,Filamentous actin ,Disasters ,Structural Biology ,Animals ,Electrophoresis, Gel, Two-Dimensional ,RNA, Messenger ,Phosphorylation ,Cytoskeleton ,Life Cycle Stages ,Dehydration ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,fungi ,Actin remodeling ,Cell Biology ,biology.organism_classification ,Actins ,Cell biology ,biology.protein ,bacteria ,Electrophoresis, Polyacrylamide Gel ,MDia1 - Abstract
Under various environmental stresses, the true slime mold Physarum polycephalum converts into dormant forms, such as microcysts, sclerotia, and spores, which can survive in adverse environments for a considerable period of time. In drought-induced sclerotia, actin is threonine phosphorylated, which blocks its ability to polymerize into filaments. It is known that fragmin and actin-fragmin kinase (AFK) mediate this phosphorylation event. In this work, we demonstrate that high levels of actin threonine phosphorylation are also found in other dormant cells, including microcysts and spores. As the threonine phosphorylation of actin in microcysts and sclerotia were induced by drought stress but not by other stresses, we suggest that drought stress is essential for actin phosphorylation in both cell types. Although characteristic filamentous actin structures (dot- or rod-like structures) were observed in microcysts, sclerotia, and spores, actin phosphorylation was not required for the formation of these structures. Prior to the formation of both microcysts and sclerotia, AFK mRNA expression was activated transiently, whereas fragmin mRNA levels decreased. Our results suggest that drought stress and AFK might be involved in the threonine phosphorylation of actin. Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc.
- Published
- 2006
33. Identification of Residues Responsible for Ligand Recognition and Regioisomeric Selectivity of Lysophosphatidic Acid Receptors Expressed in Mammalian Cells
- Author
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Gabor Tigyi, Kimiko Murakami-Murofushi, Abby L. Parrill, Akira Tokumura, Yuko Fujiwara, Daniel L. Baker, and Vineet M. Sardar
- Subjects
Models, Molecular ,Insecta ,Protein Conformation ,Stereochemistry ,Biology ,Arginine ,Ligands ,Transfection ,medicine.disease_cause ,Biochemistry ,Cell Line ,Structure-Activity Relationship ,chemistry.chemical_compound ,Cell Line, Tumor ,Lysophosphatidic acid ,medicine ,Animals ,Humans ,Gene family ,Receptors, Lysophosphatidic Acid ,Receptor ,Molecular Biology ,Gene ,Alanine ,Mutation ,Dose-Response Relationship, Drug ,Stereoisomerism ,Valine ,Cell Biology ,Hydrogen-Ion Concentration ,Flow Cytometry ,Ligand (biochemistry) ,Protein Structure, Tertiary ,Rats ,Kinetics ,Models, Chemical ,chemistry ,Mutagenesis, Site-Directed ,Calcium ,lipids (amino acids, peptides, and proteins) ,Lysophospholipids ,Baculoviridae ,Software - Abstract
The endothelial differentiation gene family encodes three highly homologous G protein-coupled receptors for lysophosphatidic acid (LPA). Based on baculoviral overexpression studies, differences have been proposed in the structure-activity relationship (SAR) of these receptors. We have compared the SAR of the individual receptors either overexpressed transiently at high or at lower levels following stable transfection in LPA-nonresponsive RH7777 cells. The SAR in transfected RH7777 cells was markedly different from that described in insect cells. The LPA(3) receptor has been proposed to be selectively activated by unsaturated LPA species and shows a strong preference for sn-2 versus the sn-1 acyl-LPA regioisomer. Because of the short half-life of sn-2 LPA due to acyl migration under some conditions, we have synthesized acyl migration-resistant analogs using an acetyl group in place of the free hydroxyl group in order to evaluate LPA receptor SAR. Only LPA(1) and LPA(2) showed regioisomeric preference and only for the 18:2 fatty acyl-stabilized LPA sn-1 regioisomer. To identify residues involved in ligand recognition of LPA(3), we developed and validated computational models of LPA(3) complexes with the analogs studied. The models revealed that Arg-3.28 and Gln-3.29 conserved within the LPA-selective endothelial differentiation gene receptors and the more variable Lys-7.35 and Arg-5.38 of LPA(3) form critical interactions with the polar headgroup of LPA. The models identified Leu-2.60 and Val-7.39 of LPA(3) underlying the regioisomer-selective interaction with the acetyl group of the stabilized regioisomers. Mutation of Leu-2.60 to alanine selectively increased the EC(50) of the sn-2 acetyl-LPA regioisomers, whereas alanine replacement of Val-7.39 profoundly affected both regioisomers.
- Published
- 2005
34. A novel pyrazolone, 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone, as a potent catalytic inhibitor of human telomerase
- Author
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Narie Sasaki, Mieko Satoh-Masuoka, Hiromu Murofushi, Yasutaka Kakiuchi, and Kimiko Murakami-Murofushi
- Subjects
Telomerase ,Deoxyribonucleosides ,Biophysics ,Pyrazolone ,Biology ,Biochemistry ,Cell Line ,Catalysis ,chemistry.chemical_compound ,Cell Line, Tumor ,medicine ,Humans ,Pyrazolones ,Molecular Biology ,IC50 ,Leukemia ,Cell Biology ,Fibroblasts ,Enzyme Activation ,Kinetics ,chemistry ,Pyrazoles ,Primer (molecular biology) ,Derivative (chemistry) ,DNA ,medicine.drug - Abstract
A new derivative of 1-phenyl-3-methyl-5-pyrazolone, 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone, named TELIN, was chemically synthesized and identified as a potent inhibitor of human telomerase in the cell-free telomeric repeat amplification protocol. TELIN inhibited telomerase activity at submicromolar level with IC50 of approximately 0.3 microM. Kinetic studies revealed that TELIN does not bind to DNA but to telomerase protein, and the mode of inhibition by this substance was competitive-noncompetitive mixed-type with respect to the TS primer, whereas it was uncompetitive or noncompetitive-uncompetitive mixed-type with respect to the three deoxyribonucleosides. These results demonstrate that TELIN is a specific potent catalytic blocker of telomerase,and is considered to be a valuable substance for medical treatment of cancer and related diseases.
- Published
- 2004
35. Transient posttranslational up-regulation of telomerase activity during megakaryocytic differentiation of K562 cells
- Author
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Mayuka Nakatake, O Yamada, Narie Sasaki, and Kimiko Murakami-Murofushi
- Subjects
Telomerase ,Base Sequence ,Chemistry ,Somatic cell ,HL60 ,Cellular differentiation ,Biophysics ,Cell Biology ,Cell cycle ,Biochemistry ,Molecular biology ,Up-Regulation ,Cell biology ,chemistry.chemical_compound ,Cell culture ,Humans ,Telomerase reverse transcriptase ,Enzyme Inhibitors ,K562 Cells ,Protein Processing, Post-Translational ,Molecular Biology ,Protein Kinase C ,DNA Primers ,K562 cells - Abstract
Telomerase is active in immature somatic cells, but not in differentiated cells. However, the mechanism by which telomerase is regulated in relation to cell differentiation is not well understood. In this study, the human erythroid leukemia cell line K562 was induced to differentiate into megakaryocytes by TPA and into erythroid by STI571. The human acute myeloblastic leukemia cell line HL60 was also induced to differentiate into monocytes by TPA. Telomerase activity, the expression of human telomerase reverse transcriptase, hTERT, and the cell cycle were examined. TPA induced a transient increase in telomerase activity during the megakaryocytic differentiation while the message of hTERT decreased gradually throughout the same period. This suggests the existence of a regulatory mechanism other than transcription of hTERT. Cell cycle analysis revealed that cells in G2/M phase increased in number in accordance with the changes in telomerase activity. Pretreatment with PKC inhibitors inhibited the megakaryocytic differentiation, transient increase in telomerase activity, and G2/M arrest. These results suggest that PKC acts as a transient post-translational activator of telomerase during megakaryocytic differentiation.
- Published
- 2004
36. Glom Is a Novel Mitochondrial DNA Packaging Protein inPhysarum polycephalumand Causes Intense Chromatin Condensation without Suppressing DNA Functions
- Author
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Shigeyuki Kawano, Tamaki Kobayashi, Yuki Shirai, Atsushi Sakai, Hiroyoshi Takano, Tetsuya Higashiyama, Haruko Kuroiwa, Chikako Nishitani, Kimiko Murakami-Murofushi, Narie Sasaki, and Tsuneyoshi Kuroiwa
- Subjects
DNA Replication ,Mitochondrial DNA ,Transcription, Genetic ,Molecular Sequence Data ,Fluorescent Antibody Technique ,Physarum polycephalum ,Biology ,DNA condensation ,DNA, Mitochondrial ,Fungal Proteins ,chemistry.chemical_compound ,Sequence Analysis, Protein ,Escherichia coli ,Animals ,Nucleoid ,Amino Acid Sequence ,Cloning, Molecular ,Microscopy, Immunoelectron ,Molecular Biology ,fungi ,DNA replication ,Articles ,Enterobacter aerogenes ,Cell Biology ,biology.organism_classification ,Molecular biology ,Chromatin ,Mitochondrial DNA packaging ,Protein Structure, Tertiary ,Cell biology ,Mitochondrial chromosome ,chemistry ,Mutation ,bacteria ,DNA - Abstract
Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions.
- Published
- 2003
37. Cyclic phosphatidic acid elicits neurotrophin-like actions in embryonic hippocampal neurons
- Author
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Tetsuyuki Kobayashi, Yuko Fujiwara, Susan O. Meakin, Agnes Sebök, Gabor Tigyi, and Kimiko Murakami-Murofushi
- Subjects
medicine.medical_specialty ,biology ,Neurite ,Phosphatidic acid ,Tropomyosin receptor kinase A ,Biochemistry ,Cell biology ,Wortmannin ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Endocrinology ,Nerve growth factor ,chemistry ,Internal medicine ,Lysophosphatidic acid ,cardiovascular system ,biology.protein ,medicine ,heterocyclic compounds ,K252a ,Neurotrophin - Abstract
Cyclic phosphatidic acid (cPA; 1-acyl-sn-glycerol-2,3-cyclic phosphate) is an analog of the growth factor-like phospholipid mediator lysophosphatidic acid (LPA). As brain tissue is the richest source of cPA we tested its effects on hippocampal neurons from day 16/17 embryonic rat cultured in a serum-free medium. Nanomolar concentrations of cPA elicited a neurotrophic effect and promoted neurite outgrowth that exceeded that of 50 ng/mL nerve growth factor (NGF). Pertussis toxin, the LPA1/LPA3 receptor-selective antagonist dioctylglycerol pyrophosphate, the myristoylated inhibitory pseudosubstrate peptide of protein kinase A (PKI), Wortmannin and PD98059 abolished the neurite-promoting effect. cPA elicited a sustained activation of extracellular signal-related kinases (ERK) 1/2 and Akt. Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, reduced cPA-induced enhancement of neurite outgrowth. In B5P cells, a clonal cell line of PC12 cells overexpressing tyrosine kinase NGF receptor (TrkA), cPA elicited transphosphorylation of TrkA. cPA-elicited ERK activation was blocked by K252a and PKI. These results suggest that cPA mimics the effects of, and activates signaling pathways similar to, the neurotrophin NGF in cultured embryonic hippocampal neurons and B5P cells.
- Published
- 2003
38. Cholesteryl Glucoside-induced Protection against Gastric Ulcer
- Author
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Shohko Kunimoto, Isao Yamatsu, Wataru Murofushi, Tetsuyuki Kobayashi, Narie Sasaki, Yukie Hasegawa, Kimiko Murakami-Murofushi, Hiromu Murofushi, and Susumu Kobayashi
- Subjects
Physiology ,Pharmacology ,Biology ,Rats, Sprague-Dawley ,Glycolipid ,Heat shock protein ,Gastric mucosa ,medicine ,Animals ,HSP70 Heat-Shock Proteins ,Stomach Ulcer ,Molecular Biology ,Molecular Structure ,Stomach ,Temperature ,Cell Biology ,General Medicine ,Lipid signaling ,Anti-Ulcer Agents ,Rats ,Hsp70 ,Heat shock factor ,Cholesterol ,medicine.anatomical_structure ,Biochemistry ,Female ,Diterpenes ,Signal transduction ,Stress, Psychological - Abstract
The cytoprotective effect of heat shock proteins (HSPs) promises new therapeutic modalities for medical treatment. We examined the anti-ulcer effect of cholesteryl glucoside (1-O-cholesteryl-beta-D-glucopyranoside, CG) on cold-restraint stress-induced gastric ulcer in rats, in terms of its correlative ability to activate heat shock factor (HSF) and to induce HSP70. Rapid induction of CG occurred in animal tissues, especially in stomach, after exposure to stress, indicating that this glycolipid might act as an anti-stress, lipid mediator involved in the very early stages of stress-induced signal transduction. Orally administered CG apparently showed anti-ulcer activity in rats via HSF activation and HSP70 induction. When compared with geranylgeranylacetone (GGA), the well known as an effective, synthetic anti-ulcer agent, CG proved to have the same level of strength on ulcer inhibition. GGA caused CG and HSP70 induction in gastric mucosa, indicating that GGA induced HSP70 via CG production. CG thus might be useful for medical treatment of stress-induced diseases, and as an anti-stress supplement for daily diet.
- Published
- 2003
39. A Novel Membrane Protein, Ros3p, Is Required for Phospholipid Translocation across the Plasma Membrane inSaccharomyces cerevisiae
- Author
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Hidemitsu Nakamura, Kimiko Murakami-Murofushi, Kazuo Emoto, Charlotta Fredriksson, Akinori Ohta, Masato Umeda, Tetsuyuki Kobayashi, Utako Kato, and Toshihide Kobayashi
- Subjects
Vesicle-associated membrane protein 8 ,Molecular Sequence Data ,Saccharomyces cerevisiae ,Peptides, Cyclic ,Sensitivity and Specificity ,Biochemistry ,Fungal Proteins ,chemistry.chemical_compound ,Gene Expression Regulation, Fungal ,Amino Acid Sequence ,Molecular Biology ,Integral membrane protein ,Phospholipids ,Phosphatidylethanolamine ,Sequence Homology, Amino Acid ,biology ,Endoplasmic reticulum ,Cell Membrane ,Membrane Proteins ,Biological Transport ,Biological membrane ,Cell Biology ,Phospholipid translocation ,Anti-Bacterial Agents ,Cell biology ,Kinetics ,Membrane glycoproteins ,chemistry ,Membrane protein ,biology.protein ,Peptides ,Sequence Alignment ,Gene Deletion - Abstract
Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Delta cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.
- Published
- 2002
40. Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological activity toward autotaxin
- Author
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Takahiro Suzuki, Ryo Tanaka, Emi Nozaki, Masaru Kato, Kimiko Murakami-Murofushi, Susumu Kobayashi, Atsuo Nakazaki, and Mari Gotoh
- Subjects
Intramolecular reaction ,Propanols ,Stereochemistry ,Clinical Biochemistry ,Phosphatidic Acids ,Pharmaceutical Science ,Biochemistry ,Chemical synthesis ,Phosphates ,Thiophosphate ,chemistry.chemical_compound ,Multienzyme Complexes ,Drug Discovery ,Humans ,Moiety ,Pyrophosphatases ,Molecular Biology ,Phosphoric Diester Hydrolases ,Organic Chemistry ,Glycidol ,Organothiophosphorus Compounds ,Biological activity ,Phosphatidic acid ,chemistry ,Phosphodiesterase I ,Epoxy Compounds ,Molecular Medicine ,Autotaxin - Abstract
The efficient synthesis of 3-O-thia-cPAs (4a-d), sulfur analogues of cyclic phosphatidic acid (cPA), has been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic thiophosphate moiety. The present synthetic route enables the synthesis of 4a-d in only four steps from the commercially available glycidol. Preliminary biological experiments showed that 4a-d exhibited a similar inhibitory effect on autotaxin (ATX) as original cPA.
- Published
- 2011
41. Ionizing radiation, inflammation, and their interactions in colon carcinogenesis in Mlh1-deficient mice
- Author
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Takamitsu Morioka, Kimiko Murakami-Murofushi, Seiji Kito, Naoki Yoshimi, Yutaka Tokairin, Tomoko Miyoshi-Imamura, Mayumi Nishimura, Benjamin J. Blyth, Shizuko Kakinuma, Mutsumi Kaminishi, Yoshiya Shimada, Shusuke Tani, and Toshiaki Kokubo
- Subjects
Cyclin-Dependent Kinase Inhibitor p21 ,Male ,Cancer Research ,Pathology ,medicine.medical_specialty ,Colorectal cancer ,Carcinogenesis ,Adenomatous Polyposis Coli Protein ,Biology ,Adenocarcinoma ,medicine.disease_cause ,MLH1 ,DNA Mismatch Repair ,Mice ,Radiation, Ionizing ,medicine ,Animals ,Colitis ,beta Catenin ,Adaptor Proteins, Signal Transducing ,Inflammation ,Mice, Knockout ,Dextran Sulfate ,Mlh1 ,Microsatellite instability ,Nuclear Proteins ,Colon carcinogenesis ,General Medicine ,Original Articles ,medicine.disease ,Ulcerative colitis ,Lynch syndrome ,digestive system diseases ,Mice, Inbred C57BL ,radiation ,Disease Models, Animal ,Oncology ,Colonic Neoplasms ,Female ,Tumor Suppressor Protein p53 ,MutL Protein Homolog 1 - Abstract
Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1(-/-) and Mlh1(+/+) mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1(+/+) mice. Colon tumors developed after DSS treatment alone in Mlh1(-/-) mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and β-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency.
- Published
- 2014
42. Cyclic phosphatidic acid treatment suppress cuprizone-induced demyelination and motor dysfunction in mice
- Author
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Yuuki Kawamura, Sosuke Yagishita, Motomu Tanaka, Takeo Awaji, Shinji Yamamoto, Kota Yamashina, Keisuke Yoshikawa, Mari Gotoh, Kei Maruyama, and Kimiko Murakami-Murofushi
- Subjects
Male ,medicine.medical_specialty ,Central nervous system ,Phosphatidic Acids ,Hippocampal formation ,Motor Activity ,chemistry.chemical_compound ,Cuprizone ,Heterocyclic Compounds, 1-Ring ,Mice ,Internal medicine ,medicine ,Demyelinating disease ,Animals ,heterocyclic compounds ,Neuroinflammation ,Pharmacology ,Multiple sclerosis ,Phosphatidic acid ,medicine.disease ,Astrogliosis ,Mice, Inbred C57BL ,Motor Skills Disorders ,Disease Models, Animal ,Endocrinology ,medicine.anatomical_structure ,Treatment Outcome ,chemistry ,cardiovascular system ,Neuroscience ,Astrocyte ,Demyelinating Diseases - Abstract
Multiple sclerosis is a chronic demyelinating disease of the central nervous system leading to progressive cognitive and motor dysfunction, which is characterized by neuroinflammation, demyelination, astrogliosis, loss of oligodendrocytes, and axonal pathologies. Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. In this study, we investigated the effects of cPA on cuprizone-induced demyelination, which is a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, astrocyte and microglial activation, and motor dysfunction. Simultaneous administration of cPA effectively attenuated cuprizone-induced demyelination, glial activation, and motor dysfunction. These data indicate that cPA may be a useful treatment to reduce the extent of demyelination and the severity of motor dysfunction in multiple sclerosis. cPA is a potential lead compound in the development of drugs for the treatment of this devastating disease.
- Published
- 2014
43. Existence of a bioactive lipid, cyclic phosphatidic acid, bound to human serum albumin
- Author
-
Tetsuyuki Kobayashi, Hiroh Ikezawa, Rieko Tanaka-Ishii, Ryo Taguchi, and Kimiko Murakami-Murofushi
- Subjects
Electrospray ionization ,Serum albumin ,Phosphatidic Acids ,Plasma protein binding ,Tandem mass spectrometry ,Mass spectrometry ,Gas Chromatography-Mass Spectrometry ,Mass Spectrometry ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,medicine ,Humans ,heterocyclic compounds ,General Pharmacology, Toxicology and Pharmaceutics ,Chromatography, High Pressure Liquid ,Serum Albumin ,chemistry.chemical_classification ,Chromatography ,biology ,Chemistry ,Fatty acid ,General Medicine ,Phosphatidic acid ,Human serum albumin ,Biochemistry ,cardiovascular system ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Chromatography, Thin Layer ,Protein Binding ,medicine.drug - Abstract
A novel bioactive lipid, cyclic phosphatidic acid (cPA), was identified in lipids bound to human serum albumin. A cPA fraction was extracted and purified from human serum albumin by use of a combination of preparative TLC and HPLC. Electrospray ionization mass spectrometry of the purified fraction showed molecular ions corresponding to cPA, which was composed of some different fatty acid species. The most abundant component was identified as palmitoyl-cPA by tandem mass spectrometry using collision-induced dissociation. These data have established that cPA is a naturally occurring lipid bound to human serum albumin.
- Published
- 1999
44. Molecular Cloning and Characterization of a Novel Human G-protein-coupled Receptor, EDG7, for Lysophosphatidic Acid
- Author
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Susumu Kobayashi, Kimiko Murakami-Murofushi, Masafumi Tsujimoto, Junken Aoki, Hiroyuki Arai, Tetsuyuki Kobayashi, Koji Bandoh, Hiroyuki Hosono, and Keizo Inoue
- Subjects
Male ,Potassium Channels ,Molecular Sequence Data ,Receptors, Cell Surface ,Spodoptera ,Biology ,Transfection ,PC12 Cells ,Biochemistry ,Cell Line ,Receptors, G-Protein-Coupled ,GTP-Binding Proteins ,Proto-Oncogene Proteins ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Receptors, Lysophosphatidic Acid ,Molecular Biology ,Phylogeny ,Unsaturated fatty acid ,DNA Primers ,ets-Domain Protein Elk-1 ,G protein-coupled receptor ,LPAR3 ,LPAR2 ,LPAR1 ,Base Sequence ,Sequence Homology, Amino Acid ,Cell Biology ,Molecular biology ,Recombinant Proteins ,Rats ,Cell biology ,DNA-Binding Proteins ,Kinetics ,Lysophospholipid receptor ,Organ Specificity ,Saturated fatty acid ,Calcium ,Female ,lipids (amino acids, peptides, and proteins) ,Lysophospholipids ,Mitogen-Activated Protein Kinases ,Autotaxin ,Sequence Alignment ,Transcription Factors - Abstract
Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation.
- Published
- 1999
45. Hyperosmotic stress-induced reorganization of actin bundles inDictyosteliumcells over-expressing cofilin
- Author
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Maiko Katadae, Masazumi Sameshima, Mikako Maruya, Hiroyuki Aizawa, Kimiko Murakami-Murofushi, and Ichiro Yahara
- Subjects
biology ,Phalloidin ,Actin remodeling ,Arp2/3 complex ,macromolecular substances ,Cell Biology ,Cofilin ,biology.organism_classification ,Actin cytoskeleton ,Dictyostelium ,Dictyostelium discoideum ,Cell biology ,chemistry.chemical_compound ,chemistry ,Genetics ,biology.protein ,Actin - Abstract
Background Cofilin is a low-molecular weight actin-modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin-interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known. Results In response to hyperosmotic stress, D. discoideum cells round up. This stress-induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction. Over-expression of cofilin increases and thickens cortical actin bundles in cells. The bundles become tight and are reorganized into a ring-shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3-nm were aligned parallel by cross-bridge structures. In cells lacking the myosin-II heavy chain, the bundles, which were induced by an over-expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over-expressing cofilin before and after exposure to hyperosmotic stress. Conclusions Cofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin-II-independent manner, in response to hyperosmotic stress.
- Published
- 1999
46. A Novel 66-kDa Stress Protein, p66, Associated with the Process of Cyst Formation of Physarum polycephalum Is a Physarum Homologue of a Yeast Actin-Interacting Protein, AIP1
- Author
-
Yukiko Shimada, Seiji Matsumoto, Mari Ogawa, Michiko Isohata, Tomoko Kasakura, Kimiko Murakami-Murofushi, Ichiro Yahara, Makoto Mitsui, and Mikako Maruya
- Subjects
DNA, Complementary ,Hot Temperature ,Molecular Sequence Data ,Physarum polycephalum ,Saccharomyces cerevisiae ,Biochemistry ,Fungal Proteins ,Complementary DNA ,Animals ,Amino Acid Sequence ,RNA, Messenger ,Northern blot ,Actin-binding protein ,Cloning, Molecular ,Molecular Biology ,Heat-Shock Proteins ,Actin ,Messenger RNA ,Base Sequence ,Sequence Homology, Amino Acid ,Physarum ,biology ,Chemistry ,Microfilament Proteins ,fungi ,General Medicine ,biology.organism_classification ,Actins ,Yeast ,Cell biology ,biology.protein - Abstract
When exposed to various stresses including heat shock, myxoamoebae, growing haploid cells of Physarum polycephalum, show marked morphological changes and consequently become disk-shaped microcysts. We have found that p66 is induced exclusively in the course of microcyst formation and has an actin-binding activity. In this study, we purified p66 to homogeneity and isolated a p66 cDNA. The deduced protein sequence contained 601 amino acids and showed 31% identity to a yeast actin-interacting protein, AIP1. Northern blot analysis revealed that the amount of p66 mRNA was significantly increased by heat shock in myxoamoebae but not in plasmodia. Thus, p66 seems to be a developmentally-expressed stress protein which regulates the rearrangement of actin organization during microcyst formation in P. polycephalum.
- Published
- 1998
47. Characterization of Endogenous and Heterologously Expressed LPA Receptor Subtypes
- Author
-
David J. Fischer, Kimiko Murakami-Murofushi, and Gabor Tigyi
- Subjects
Chemistry ,General Neuroscience ,Receptors, Cell Surface ,Endogeny ,Culture Media, Serum-Free ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Receptors, G-Protein-Coupled ,Cell biology ,History and Philosophy of Science ,Animals ,Humans ,Receptors, Lysophosphatidic Acid ,Receptor - Published
- 2006
48. Cholesterol glucosylation is catalyzed by transglucosylation reaction of β-glucosidase 1
- Author
-
Hisako Akiyama, Yoshio Hirabayashi, Kimiko Murakami-Murofushi, and Susumu Kobayashi
- Subjects
Glycosylation ,Cholesteryl glucoside ,Biophysics ,Gaucher disease ,Glycolipid ,Biology ,Biochemistry ,Catalysis ,Cell Line ,chemistry.chemical_compound ,β-Glucosidase 1 (GBA1) ,Mice ,GBA3 ,Animals ,Humans ,HSF1 ,Molecular Biology ,chemistry.chemical_classification ,Cholesterol ,beta-Glucosidase ,Fatty acid ,Sterol glucosyltransferase ,Cell Biology ,Molecular biology ,Hsp70 ,Heat shock factor ,Enzyme ,chemistry ,Transglucosylation ,Saturated fatty acid ,Glucosylceramidase - Abstract
Cholesteryl glucoside (β-ChlGlc), a monoglucosylated derivative of cholesterol, is involved in the regulation of heat shock responses. β-ChlGlc, which is rapidly induced in response to heat shock, activates heat shock transcription factor 1 (HSF1) leading to the expression of heat shock protein 70 (HSP70) in human fibroblasts. Identification and biochemical characterization of the enzyme responsible for β-ChlGlc formation is important for a complete understanding of the molecular mechanisms leading to HSP70-induction following heat shock. Recently, we demonstrated that β-ChlGlc synthesis is not dependent on UDP-Glucose but glucosylceramide (GlcCer) in animal tissue and human fibroblasts. In this study, we examined the possibility of glucocerebrosidase, a GlcCer-degrading glycosidase, acting as β-ChlGlc-synthesizing enzyme. Overexpression of β-glucosidase 1 (GBA1, lysosomal acid β-glucocerebrosidase) led to an increase in cholesterol glucosylation activity in human fibroblasts. Using a cell line generated from type 2 Gaucher disease patients with severe defects in GBA1 activity, we found that cholesterol glucosylation activity was very low in the cells and the overexpression of GBA1 rescued the activity. In addition, purified recombinant GBA1 exhibits conduritol B-epoxide-sensitive cholesterol glucosylation activity. The optimum pH and temperature for cholesterol glucosylation by GBA1 were at about 5.3 and 43 °C, respectively. Short chain C8:0-GlcCer was the most effective donor for cholesterol glucosylation activity among GlcCer containing saturated fatty acid (C8:0 to C18:0) tested. GlcCer containing mono-unsaturated fatty acid was more preferred substrate for cholesterol glucosylation when compared with GlcCer containing same chain length of saturated fatty acid. These results demonstrate, for the first time, a novel function of GBA1 as a β-ChlGlc-synthesizing enzyme. Therefore, our results also reveal a new pathway for glycolipid metabolism in mammals.
- Published
- 2013
49. The macroscopic structure of RADA16 peptide hydrogel stimulates monocyte/macrophage differentiation in HL60 cells via cholesterol synthesis
- Author
-
Kimiko Murakami-Murofushi, Noritaka Hirohashi, and Yasutaka Kakiuchi
- Subjects
Scaffold ,Cellular differentiation ,Molecular Sequence Data ,Biophysics ,Molecular Conformation ,Gene Expression ,Peptide ,HL-60 Cells ,Biology ,Biochemistry ,Monocytes ,Tissue engineering ,Gene expression ,Sense (molecular biology) ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,Tissue Engineering ,Tissue Scaffolds ,Macrophages ,Biological Transport ,Cell Differentiation ,Hydrogels ,Cell Biology ,Cell biology ,Cholesterol ,chemistry ,Peptides ,Intracellular ,Biomarkers ,Signal Transduction - Abstract
Cells can sense physical properties of surrounding 3-dimensional (3D) culture substrata; however, the physiological influences of such sensing are not fully understood. Here, we studied the physiological characteristics and activities of the macroscopic structure of a routinely used 3D culture substrata, the RADA16 self-assembling peptide scaffold. We found that RADA16 exhibited three distinct assembly patterns depending on its concentration, and one of these assemblies, formed with 0.01% (w/v) RADA16, was capable of inducing differentiation of human myelocytic leukemia HL-60 cells into monocytes/macrophages. This activity was largely reduced by destroying the 3D structure of the assembly, suggesting that the assembly intrinsically retained the ability to induce HL-60 differentiation. When cultured in the RADA16 scaffold, HL-60 cells accumulated intracellular cholesterol about 10 times more than normally cultured cells. Both the RADA16 culture and cholesterol loading brought about similar gene expression profiles. These results showed that HL-60 cells can sense the physical properties of the RADA16 scaffold through a mechanism that may involve intracellular pathways of cholesterol synthesis and/or transport.
- Published
- 2013
50. Physarolisin
- Author
-
Wataru Nishii, Kimiko Murakami-Murofushi, and Kenji Takahashi
- Subjects
Chemistry - Published
- 2013
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