34 results on '"Tetsuyuki Kobayashi"'
Search Results
2. Erylysin A inhibits cytokinesis in Escherichia coli by binding with cardiolipin
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Rika Serizawa, Tetsuyuki Kobayashi, Hikari Uzawa, Tomoko Sakihara, and Naoko Takiguchi
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Pterocarpans ,Cardiolipins ,Cell ,Lipid Bilayers ,Mitochondrion ,Biochemistry ,chemistry.chemical_compound ,medicine ,Cardiolipin ,Escherichia coli ,Lipid bilayer ,Cytoskeleton ,Molecular Biology ,Cytokinesis ,Chemistry ,Escherichia coli Proteins ,Cell Membrane ,Membrane structure ,General Medicine ,Cell biology ,Mitochondria ,Cytoskeletal Proteins ,medicine.anatomical_structure ,Membrane curvature ,Cell Division - Abstract
Cardiolipin (CL) localizes to curved membranes such as cristae in mitochondria as well as cell poles and division sites in rod-shaped bacteria. CL is believed to stabilize the membrane curvature by localizing to sites of negative curvature. However, this hypothesis has not been tested because of a lack of appropriate tools to distinguish CL inside and outside lipid bilayers. In this study, we provided the first evidence that CL localized to regions of negative curvature in Escherichia coli using the novel CL probe erylysin A-EGFP (EryA-EGFP). Staining in E.coli illustrated that CL localized to the inner leaflets at cell poles and the outer leaflets at division sites. Furthermore, we revealed that EryA-EGFP inhibited cytokinesis. We propose that cytokinesis completes after CL in the outer leaflets transfers to the inner leaflets at division sites by inspecting the mechanism of inhibition of cytokinesis. Moreover, the cytoskeletal protein RodZ was abnormally distributed when cytokinesis was inhibited by EryA-EGFP, suggesting that RodZ participates in cytokinesis. In summary, we revealed the detailed distribution of CL and proposed a new model of cytokinesis.
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- 2021
3. 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
4. A novel sphingomyelin/cholesterol domain‐specific probe reveals the dynamics of the membrane domains during virus release and in Niemann‐Pick type C
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Kozo Nishibori, Rinshi S. Kasai, Shota Sakai, Gregor Anderluh, Tetsuyuki Kobayashi, Shuku Kubo, Chan-Gi Pack, Yukiko Shimada, Peter Greimel, Yasushi Sako, Motohide Murate, Mitsuhiro Abe, Takehiko Inaba, Takanori Kigawa, Fumihiro Fujimori, Françoise Hullin-Matsuda, Naoya Tochio, Makoto Yamashita, Takuma Kishimoto, Naoshi Dohmae, Yoko Aida, Hideyuki Miyatake, Kyoji Hagiwara, Asami Makino, Robert G. Parton, Yutaka Sasaki, Toshihide Kobayashi, Reiko Ishitsuka, Atsushi Kurahashi, Akihiro Kusumi, Nicole L. Schieber, Hideko Tanaka, Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL)
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0301 basic medicine ,[SDV]Life Sciences [q-bio] ,Membrane lipids ,Cell ,Biology ,Biochemistry ,Virus ,Fungal Proteins ,03 medical and health sciences ,Membrane Microdomains ,Genetics ,medicine ,Humans ,Small GTPase ,Molecular Biology ,Lipid raft ,Cells, Cultured ,Virus Release ,Binding Sites ,030102 biochemistry & molecular biology ,Niemann-Pick Disease, Type C ,Fibroblasts ,Sphingolipid ,Sphingomyelins ,3. Good health ,Cell biology ,Cholesterol ,030104 developmental biology ,medicine.anatomical_structure ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin ,Grifola ,HeLa Cells ,Protein Binding ,Biotechnology - Abstract
International audience; We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.
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- 2016
5. Novel role of group VIB Ca2+-independent phospholipase A2γ in leukocyte-endothelial cell interactions
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Junichi Aiboshi, Masahiro Shibata, Yasuhiro Otomo, Tetsuyuki Kobayashi, and Mitsuaki Kojima
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Male ,Neutrophils ,Leukocyte Rolling ,Cell Communication ,Critical Care and Intensive Care Medicine ,Sensitivity and Specificity ,Random Allocation ,Phospholipase A2 ,Cell Movement ,In vivo ,Animals ,Humans ,Macrophage ,Medicine ,Mesentery ,Phospholipid Transfer Proteins ,Rats, Wistar ,Cells, Cultured ,biology ,business.industry ,Role ,Degranulation ,Endothelial Cells ,hemic and immune systems ,Chemotaxis ,Molecular biology ,Rats ,Endothelial stem cell ,Chemotaxis, Leukocyte ,Disease Models, Animal ,Phospholipases A2, Calcium-Independent ,Immunology ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Surgery ,business ,Intravital microscopy - Abstract
BACKGROUND Phospholipase A2 (PLA2) is associated with a variety of inflammatory processes related to polymorphonuclear neutrophil (PMN)-endothelial cell interactions. However, the cellular and molecular mechanisms underlying the interactions and the causative isoform(s) of PLA2 remain elusive. In addition, we recently showed that calcium-independent PLA2γ (iPLA2γ), but not cytosolic PLA2 (cPLA2), is responsible for the cytotoxic functions of human PMN including respiratory bursts, degranulation, and chemotaxis. We therefore hypothesized that iPLA2γ is a prerequisite for the PMN recruitment cascade into the site of inflammation. The aim of this study was to elucidate the roles of the three major phospholipases A2, iPLA2, cPLA2 and secretory PLA2, in leukocyte rolling and adherence and in the surface expression of β2-integrins in vivo and in vitro in response to well-defined stimuli. METHODS Male Wistar rats were pretreated with PLA2 inhibitors selective for iPLA2β, iPLA2γ, cPLA2, or secretory PLA2. Leukocyte rolling/adherence in the mesenteric venules superfused with platelet-activating factor (PAF) were quantified by intravital microscopy. Furthermore, isolated human PMNs or whole blood were incubated with each PLA2 inhibitor and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or PAF. PMN adherence was assessed by counting cells bound to purified fibrinogen, and the surface expression of lymphocyte function-associated antigen 1 and macrophage antigen 1 (Mac-1) was measured by flow cytometry. RESULTS The iPLA2γ-specific inhibitor almost completely inhibited the fMLP/PAF-induced leukocyte adherence in vivo and in vitro and also decreased the fMLP/PAF-stimulated surface expression of Mac-1 by 60% and 95%, respectively. In contrast, the other inhibitors did not affect these cellular functions. CONCLUSION iPLA2γ seems to be involved in leukocyte/PMN adherence in vivo and in vitro as well as in the up-regulation of Mac-1 in vitro in response to PAF/fMLP. This enzyme is therefore likely to be a major regulator in the PMN recruitment cascade.
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- 2015
6. The Adipocyte-Inducible Secreted Phospholipases PLA2G5 and PLA2G2E Play Distinct Roles in Obesity
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Hiroyasu Sato, Tetsuya Hirabayashi, Yuki Isogai, Michael H. Gelb, Tetsuyuki Kobayashi, Yuichi Oike, Kei Yamamoto, Takumi Kojima, Yasumasa Nishito, Yoshitaka Taketomi, Shuntaro Hara, Satoshi Ida, Yuji Miyamoto, Ayako Ushida, Yoshimi Miki, Makoto Murakami, Masayuki Watanabe, Ryo Taguchi, Hideo Baba, Keishi Miyata, and Kazutaka Ikeda
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Leptin ,medicine.medical_specialty ,Time Factors ,Physiology ,Adipose Tissue, White ,Lipoproteins ,Macrophage polarization ,Mice, Obese ,Adipose tissue ,Phospholipase ,Biology ,Diet, High-Fat ,Group II Phospholipases A2 ,Article ,Group V Phospholipases A2 ,Mice ,chemistry.chemical_compound ,Insulin resistance ,Adipocyte ,Internal medicine ,medicine ,Animals ,Humans ,Insulin ,Obesity ,RNA, Messenger ,Molecular Biology ,Cells, Cultured ,Inflammation ,Mice, Knockout ,Phosphatidylethanolamine ,Macrophages ,Lipid metabolism ,Cell Biology ,Glucose Tolerance Test ,medicine.disease ,Mice, Inbred C57BL ,Endocrinology ,Liver ,chemistry ,Female ,Proto-Oncogene Proteins c-akt ,Lipoprotein - Abstract
Metabolic disorders including obesity and insulin resistance have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5−/− and Pla2g2e−/− mice revealed distinct and previously unrecognized roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia and obesiy. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of “metabolic sPLA2s” adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.
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- 2014
7. Group X Secreted Phospholipase A2 Releases ω3 Polyunsaturated Fatty Acids, Suppresses Colitis, and Promotes Sperm Fertility*
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Yasumasa Nishito, Ayako Ushida, Hiroyasu Sato, Remi Murase, Toshinori Yamamoto, Makoto Murakami, Kazutaka Ikeda, Kei Yamamoto, Tetsuyuki Kobayashi, and Yoshitaka Taketomi
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0301 basic medicine ,Male ,medicine.medical_specialty ,Colon ,Prostaglandin ,Gene Expression ,Mice, Transgenic ,Biology ,Biochemistry ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Phospholipase A2 ,Internal medicine ,Fatty Acids, Omega-6 ,Fatty Acids, Omega-3 ,medicine ,Animals ,Group X Phospholipases A2 ,Humans ,Transgenes ,Colitis ,Molecular Biology ,chemistry.chemical_classification ,Phospholipase A ,Arachidonic Acid ,Sperm Count ,Gene Expression Profiling ,Dextran Sulfate ,Interleukin-17 ,Fatty acid ,GPR120 ,Cell Biology ,medicine.disease ,Lipids ,Spermatozoa ,Phospholipases A2 ,030104 developmental biology ,Endocrinology ,Fertility ,chemistry ,biology.protein ,Sperm Motility ,Th17 Cells ,Arachidonic acid ,Polyunsaturated fatty acid - Abstract
Within the secreted phospholipase A2 (sPLA2) family, group X sPLA2 (sPLA2-X) has the highest capacity to hydrolyze cellular membranes and has long been thought to promote inflammation by releasing arachidonic acid, a precursor of pro-inflammatory eicosanoids. Unexpectedly, we found that transgenic mice globally overexpressing human sPLA2-X (PLA2G10-Tg) displayed striking immunosuppressive and lean phenotypes with lymphopenia and increased M2-like macrophages, accompanied by marked elevation of free ω3 polyunsaturated fatty acids (PUFAs) and their metabolites. Studies using Pla2g10-deficient mice revealed that endogenous sPLA2-X, which is highly expressed in the colon epithelium and spermatozoa, mobilized ω3 PUFAs or their metabolites to protect against dextran sulfate-induced colitis and to promote fertilization, respectively. In colitis, sPLA2-X deficiency increased colorectal expression of Th17 cytokines, and ω3 PUFAs attenuated their production by lamina propria cells partly through the fatty acid receptor GPR120. In comparison, cytosolic phospholipase A2 (cPLA2α) protects from colitis by mobilizing ω6 arachidonic acid metabolites, including prostaglandin E2. Thus, our results underscore a previously unrecognized role of sPLA2-X as an ω3 PUFA mobilizer in vivo, segregated mobilization of ω3 and ω6 PUFA metabolites by sPLA2-X and cPLA2α, respectively, in protection against colitis, and the novel role of a particular sPLA2-X-driven PUFA in fertilization.
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- 2016
8. Lipid Polarity Is Maintained in Absence of Tight Junctions
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Kazutaka Ikeda, Satoshi B. Sato, Mayu Suzuki, Tetsuyuki Kobayashi, Donna B. Stolz, Ryo Taguchi, Junichi Ikenouchi, Kazuaki Umeda, Toshihide Kobayashi, and Masato Umeda
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Tight junction ,Membrane lipids ,Cell Membrane ,Cell Polarity ,Epithelial Cells ,Cell Biology ,Biology ,Apical membrane ,Biochemistry ,Cell Line ,Sphingomyelins ,Tight Junctions ,Cell biology ,Cell membrane ,medicine.anatomical_structure ,Cell polarity ,Zonula Occludens Proteins ,medicine ,Humans ,Sphingomyelin ,Molecular Biology ,Epithelial polarity - Abstract
The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.
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- 2012
9. Physiological Roles of Group X-secreted Phospholipase A2 in Reproduction, Gastrointestinal Phospholipid Digestion, and Neuronal Function
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Yoshikazu Ishimoto, Yuki Isogai, Kohji Hanasaki, Shuntaro Hara, Makoto Murakami, Noriko Suzuki, Yoshitaka Taketomi, Yukio Ishikawa, Seiko Masuda, Yasunori Yokota, Kazutaka Ikeda, Hiroyasu Sato, Kei Yamamoto, Yoshimi Miki, Daisuke Kamei, Toshiharu Ishii, Ryo Taguchi, Toshiko Suzuki-Yamamoto, and Tetsuyuki Kobayashi
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Male ,Nervous system ,medicine.medical_specialty ,Transgene ,Inflammation ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Mice ,Phospholipase A2 ,Internal medicine ,medicine ,Animals ,Phospholipases A2, Secretory ,Molecular Biology ,Phospholipids ,Mice, Knockout ,Neurons ,Gastrointestinal tract ,Phospholipase A ,biology ,Reproduction ,Cell Biology ,Lipids ,Gastrointestinal Tract ,Endocrinology ,medicine.anatomical_structure ,Eicosanoid ,Organ Specificity ,biology.protein ,Digestion ,Female ,medicine.symptom ,Homeostasis - Abstract
Although the secreted phospholipase A(2) (sPLA(2)) family has been generally thought to participate in pathologic events such as inflammation and atherosclerosis, relatively high and constitutive expression of group X sPLA(2) (sPLA(2)-X) in restricted sites such as reproductive organs, the gastrointestinal tract, and peripheral neurons raises a question as to the roles played by this enzyme in the physiology of reproduction, digestion, and the nervous system. Herein we used mice with gene disruption or transgenic overexpression of sPLA(2)-X to clarify the homeostatic functions of this enzyme at these locations. Our results suggest that sPLA(2)-X regulates 1) the fertility of spermatozoa, not oocytes, beyond the step of flagellar motility, 2) gastrointestinal phospholipid digestion, perturbation of which is eventually linked to delayed onset of a lean phenotype with reduced adiposity, decreased plasma leptin, and improved muscle insulin tolerance, and 3) neuritogenesis of dorsal root ganglia and the duration of peripheral pain nociception. Thus, besides its inflammatory action proposed previously, sPLA(2)-X participates in physiologic processes including male fertility, gastrointestinal phospholipid digestion linked to adiposity, and neuronal outgrowth and sensing.
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- 2011
10. Hair Follicular Expression and Function of Group X Secreted Phospholipase A2 in Mouse Skin
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Yoshitaka Taketomi, Hiroki Nakanishi, Toshiharu Ishii, Yoshimi Miki, Yoshikazu Ishimoto, Yukio Ishikawa, Kazutaka Ikeda, Kiyoko Fukami, Yuki Isogai, Noriko Suzuki, Yasumasa Nishito, Kohji Hanasaki, Makoto Murakami, Kei Yamamoto, Tetsuyuki Kobayashi, Seiko Masuda, Ryo Taguchi, Yasunori Yokota, Kiyokazu Morioka, and Hiroyasu Sato
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medicine.medical_specialty ,Biology ,Outer root sheath ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Melanin ,Mice ,Phospholipase A2 ,Hair cycle ,Internal medicine ,medicine ,Animals ,Homeostasis ,Phospholipases A2, Secretory ,Molecular Biology ,Melanins ,Mice, Knockout ,Phospholipase A ,integumentary system ,Epidermis (botany) ,Alopecia ,Cell Biology ,Hair follicle ,Lipids ,Epithelium ,Enzyme Activation ,medicine.anatomical_structure ,Endocrinology ,Fatty Acids, Unsaturated ,Phosphatidylcholines ,Prostaglandins ,biology.protein ,sense organs ,Hair Follicle - Abstract
Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A(2) (PLA(2)) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA(2) (sPLA(2)-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA(2)-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA(2)-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA(2)-X in hair follicles, the presence of skin-specific machinery leading to sPLA(2)-X activation, a functional link of sPLA(2)-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis.
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- 2011
11. 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
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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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12. Group III secreted phospholipase A2 transgenic mice spontaneously develop inflammation
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Kei Yamamoto, Makoto Murakami, Yuki Isogai, Hiroyasu Sato, Seiko Masuda, Tetsuyuki Kobayashi, and Yoshitaka Taketomi
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HDC, histidine decarboxylase ,Chemokine ,LNL, LoxP–neomycin-resistance gene–LoxP ,MPO, myeloperoxidase ,Dermatitis ,Acanthosis ,Biochemistry ,PC, phosphatidylcholine ,Mice ,chemistry.chemical_compound ,LDL, low-density lipoprotein ,Prostaglandin E2 ,mPGES, microsomal prostaglandin E synthase ,Skin ,ESI, electrospray ionization ,RT, reverse transcription ,Group III Phospholipases A2 ,MCP-1, monocyte chemoattractant protein-1 ,GAPDH, glyceraldehyde-3-phosphate dehydrogenase ,TNFα, tumour necrosis factor α ,LPS, lipopolysaccharide ,prostaglandin ,medicine.symptom ,NK, natural killer ,Research Article ,medicine.drug ,Genetically modified mouse ,LTB4, leukotriene B4 ,HDL, high-density lipoprotein ,Prostaglandin ,Mice, Transgenic ,Inflammation ,CAG, chicken β-actin ,Biology ,Gene Expression Regulation, Enzymologic ,COX, cyclo-oxygenase ,PAS, periodic acid–Schiff ,sPLA2, secretory PLA2 ,Tg, transgenic ,PLA2G2A (etc.), sPLA2 group IIA (etc.) ,PGE2, prostaglandin E2 ,medicine ,Animals ,IFN, interferon ,cPLA2, cytosolic PLA2 ,mMCP, mouse mast cell protease ,PAF, platelet-activating factor ,Parakeratosis ,Molecular Biology ,phospholipid ,splenomegaly ,PLA2, phospholipase A2 ,Cell Biology ,BRAK, breast- and kidney-expressed chemokine ,medicine.disease ,WT, wild-type ,Sialadenitis ,IL, interleukin ,transgenic mouse ,chemistry ,Immunology ,biology.protein ,phospholipase A2 - Abstract
PLA2 (phospholipase A2) group III is an atypical sPLA2 (secretory PLA2) that is homologous with bee venom PLA2 rather than with other mammalian sPLA2s. In the present paper, we show that endogenous group III sPLA2 (PLA2G3) is expressed in mouse skin and that Tg (transgenic) mice overexpressing human PLA2G3 spontaneously develop skin inflammation. Pla2g3-Tg mice over 9 months of age frequently developed dermatitis with hyperkeratosis, acanthosis, parakeratosis, erosion, ulcer and sebaceous gland hyperplasia. The dermatitis was accompanied by infiltration of neutrophils and macrophages and by elevated levels of pro-inflammatory cytokines, chemokines and prostaglandin E2. In addition, Pla2g3-Tg mice had increased lymph aggregates and mucus in the airway, lymphocytic sialadenitis, hepatic extramedullary haemopoiesis, splenomegaly with increased populations of granulocytes and monocytes/macrophages, and increased serum IgG1. Collectively, these observations provide the first demonstration of spontaneous development of inflammation in mice with Tg overexpression of mammalian sPLA2.
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- 2009
13. Analyses of Group III Secreted Phospholipase A2 Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis
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Hiroyasu Sato, Rina Kato, Kazutaka Ikeda, Yukio Ishikawa, Seiko Masuda, Shuntaro Hara, Joe Yamada, Makoto Murakami, Yoshitaka Taketomi, Yuki Isogai, Shinji Hatakeyama, Kae Tsutsumi, Ryo Taguchi, Mitsuhiro Ohtsuki, Kei Yamamoto, Go-ichi Saka, Toshiharu Ishii, Hiroyuki Itabe, Tetsuyuki Kobayashi, and Ichiro Kudo
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Genetically modified mouse ,medicine.medical_specialty ,Transgene ,Mice, Transgenic ,Lipids and Lipoproteins: Metabolism, Regulation, and Signaling ,Biochemistry ,Substrate Specificity ,Apolipoproteins E ,Mice ,chemistry.chemical_compound ,Phospholipase A2 ,In vivo ,Internal medicine ,medicine ,Animals ,Molecular Biology ,Foam cell ,Sequence Homology, Amino Acid ,biology ,Group III Phospholipases A2 ,Lysophosphatidylcholines ,Cell Biology ,Atherosclerosis ,Protein Structure, Tertiary ,Isoenzymes ,Lipoproteins, LDL ,Bee Venoms ,Endocrinology ,Lysophosphatidylcholine ,chemistry ,Phosphatidylcholines ,biology.protein ,Diet, Atherogenic ,lipids (amino acids, peptides, and proteins) ,Lipoproteins, HDL ,Protein Processing, Post-Translational ,Ex vivo ,Foam Cells - Abstract
Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.
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- 2008
14. Cyclic Phosphatidic Acid Is Produced by Autotaxin in Blood
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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
15. 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
16. 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
17. Separation and Characterization of Late Endosomal Membrane Domains
- Author
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Julien Chevallier, Pierre Cosson, Jean Gruenberg, Asami Makino, Nathalie Mayran, Cécile Lebrand, Toshihide Kobayashi, Jean-Michel Escola, Tetsuyuki Kobayashi, and Marie-Hélène Beuchat
- Subjects
Endosome ,Membrane lipids ,education ,Endocytic cycle ,Population ,Endosomes ,Fatty Acids, Nonesterified ,Biology ,Kidney ,Membrane Fusion ,Biochemistry ,Cell Line ,Membrane Lipids ,Cricetinae ,Organelle ,Animals ,Molecular Biology ,Phospholipids ,education.field_of_study ,Lipid bilayer fusion ,Biological membrane ,Intracellular Membranes ,Cell Biology ,Hydrogen-Ion Concentration ,Cell biology ,Membrane ,Monoglycerides ,Lysophospholipids - Abstract
Very little is known about the biophysical properties and the lipid or protein composition of membrane domains presumably present in endocytic and biosynthetic organelles. Here we analyzed the membrane composition of late endosomes by suborganellar fractionation in the absence of detergent. We found that the internal membranes of this multivesicular organelle can be separated from the limiting membrane and that each membrane population exhibited a defined composition. Our data also indicated that internal membranes may consist of at least two populations, containing primarily phosphatidylcholine or lysobisphosphatidic acid as major phospholipid, arguing for the existence of significant microheterogeneity within late endosomal membranes. We also found that lysobisphosphatidic acid exhibited unique pH-dependent fusogenic properties, and we speculated that this lipid is an ideal candidate to regulate the dynamic properties of this internal membrane mosaic.
- Published
- 2002
18. Multiple Mechanisms Linked to Platelet Activation Result in Lysophosphatidic Acid and Sphingosine 1-Phosphate Generation in Blood
- Author
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Atsushi Wada, Gabor Tigyi, Tetsuyuki Kobayashi, Yutaka Yatomi, Takamitsu Sano, Yasuyuki Igarashi, Daniel L. Baker, and Tamas Virag
- Subjects
Blood Platelets ,Lipopolysaccharides ,Time Factors ,Xenopus ,Models, Biological ,Biochemistry ,Mass Spectrometry ,Phospholipases A ,chemistry.chemical_compound ,Sphingosine ,Lysophosphatidic acid ,Animals ,Humans ,Platelet ,Platelet activation ,Blood Coagulation ,Molecular Biology ,Phosphatidylethanolamine ,Phosphoric Diester Hydrolases ,Chemistry ,Thrombin ,Cell Biology ,Phosphatidylserine ,Lipid Metabolism ,Platelet Activation ,Lipids ,Phospholipases A1 ,Phospholipases A2 ,Lysophospholipase ,Oocytes ,Biological Assay ,Calcium ,lipids (amino acids, peptides, and proteins) ,Chromatography, Thin Layer ,Chlorine ,Lysophospholipids ,biological phenomena, cell phenomena, and immunity ,Autotaxin ,Chromatography, Liquid - Abstract
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [32P]orthophosphate, and the production of [32P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from platelets in vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA1 and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A1 and A2 and then to LPA by plasma lysophospholipase D.
- Published
- 2002
19. 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
20. Effects of Docosahexaenoic and Arachidonic Acids on the Synthesis and Distribution of Aminophospholipids during Neuronal Differentiation of PC12 Cells
- Author
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Harumi Okuyama, Atsushi Ikemoto, Masato Umeda, Kazuo Emoto, Tetsuyuki Kobayashi, and Shiro Watanabe
- Subjects
Docosahexaenoic Acids ,Neurite ,Biophysics ,Phosphatidylserines ,Biology ,Tritium ,PC12 Cells ,Peptides, Cyclic ,Biochemistry ,Serine ,Hemolysin Proteins ,chemistry.chemical_compound ,Ethanolamine ,Animals ,Nerve Growth Factors ,Molecular Biology ,Phospholipids ,Cell Size ,Neurons ,Phosphatidylethanolamine ,Arachidonic Acid ,Phosphatidylethanolamines ,Cell Membrane ,Cell Differentiation ,Phosphatidylserine ,Flow Cytometry ,Anti-Bacterial Agents ,Rats ,Nerve growth factor ,Trinitrobenzenesulfonic Acid ,chemistry ,Docosahexaenoic acid ,Arachidonic acid ,Peptides - Abstract
We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.
- Published
- 1999
21. Assessment of the possible adverse effects of oils enriched with n-3 fatty acids in rats: peroxisomal proliferation, mitochondrial dysfunctions and apoplexy
- Author
-
Tetsuyuki Kobayashi, Yuji Fukamizu, Tetsuya Shimizugawa, Harumi Okuyama, Shiro Watanabe, and Min-Zhao Huang
- Subjects
Nutrition and Dietetics ,food.ingredient ,Linolenic acid ,Endocrinology, Diabetes and Metabolism ,Linoleic acid ,Clinical Biochemistry ,Biology ,Fish oil ,Biochemistry ,Perilla oil ,Eicosapentaenoic acid ,Soybean oil ,chemistry.chemical_compound ,food ,chemistry ,Docosahexaenoic acid ,Food science ,Molecular Biology ,Unsaturated fatty acid - Abstract
Possible adverse effects of excess intake of dietary n-3 fatty acids have been reevaluated in rats by measuring peroxisomal proliferation, mitochondrial enzyme activity and incidence of apoplexy. When Sprague-Dawley rats were fed diets containing either fish oil rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or perilla seed oil rich in α-linolenic acid for 4 or 12 weeks, DHA accumulated in phospholipids of liver and heart in the fish oil group, but not in the perilla oil group when compared with safflower and soybean oil groups. Feeding a diet containing 15 weight % of fish oil induced a significant proliferation of peroxisomes compared with safflower oil-diet, but the proliferating activity of perilla oil was much less. The peroxisomal β-oxidation activities were negatively correlated with neutral lipid contents in liver. Heart mitochondrial cytochrome c oxidase activity was not affected significantly by feeding fish oil or perilla oil in the presence of an essential amount of linoleic acid for up to 12 weeks, though the proportion of linoleic acid in heart cardiolipin decreased from >86% in the safflower and soybean oil groups to 81% in the perilla oil group and to 33% in the fish oil group. Feeding n-3-enriched fish oil and perilla oil in 10 weight % of the diets did not accelerate the onset of cerebral bleeding in stroke-prone spontaneously hypertensive rats (SHRSP). Although the proliferating activity of peroxisomes by excess intake of fish oils should be noticed, these results provide evidence that n-3-enriched oils are safe under conditions applicable to human nutrition, considering the levels fed the rats were several fold higher than the anticipated maximal intake in human.
- Published
- 1996
22. Mitogenic response of near-diploid mouse cell line m5S/1M induced by epidermal growth factor
- Author
-
Hiroshi Utsumi, Masao S. Sasaki, Hitoshi Arita, Tohoru Nakano, Sachihiko Watanabe, Keizo Inoue, Masato Umeda, Tetsuyuki Kobayashi, and Tomoko Nomura
- Subjects
Cell division ,Fura-2 ,Physiology ,Inositol Phosphates ,Clinical Biochemistry ,Mitosis ,Palmitic Acids ,Biology ,Glycosphingolipids ,Cell Line ,Mice ,chemistry.chemical_compound ,Epidermal growth factor ,Animals ,Inositol ,Carbon Radioisotopes ,Receptor ,Epidermal Growth Factor ,Globosides ,Cell growth ,Trihexosylceramides ,Cell Biology ,Fibroblasts ,Diploidy ,Molecular biology ,chemistry ,Cell culture ,Calcium ,Cell Division ,hormones, hormone substitutes, and hormone antagonists ,Intracellular - Abstract
A nonmalignant near-diploid cell line m5s/1M, established by Sasaki and Kodama (J. Cell. Res., 131:114-122, 1987), was shown to respond to the epidermal growth factor (EGF). The m5s/1M cells showed high sensitivity to post-confluence inhibition of cell division and formed a uniform monolayer after the cells had become confluent. The addition of EGF resulted in loss of contact-dependent inhibition of growth and caused a massive piling up of a multilayered array of cells after they had become confluent. When EGF was removed from the medium, the cell number decreased rapidly, and the cells formed a uniform monolayer at the density observed in the absence of EGF. m5S/1M cells have high- and low-affinity receptors for EGF (approximately 40,000 receptors per cell), and the apparent dissociation constants of the EGF-binding reactions were 3.3 nM and 0.15 nM, respectively. The effect of EGF on the intracellular mobilization of Ca2+ and the formation of inositol phosphates was studied by using the calcium-sensitive fluorescent indicator fura 2 and [3H]inositol. EGF had no effect either on the mobilization of cytosolic free calcium [( Ca2+]i) or on the formation of inositol phosphates in m5s/1M cells, whereas bradykinin induced a rapid increase in both [Ca2+]i and inositol phosphates. Analysis of the glycosphingolipid (GSL) composition of m5S/1M cells showed that globotriaosylceramide (Gb3Cer), which is known to be a Burkitt lymphoma-associated antigen, is specifically expressed in the EGF-treated cells. The expression of Gb3Cer is dependent on the presence of EGF, with a reversible shift in GSL composition being observed in the presence or absence of EGF.
- Published
- 1990
23. Inhibition of transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic phosphatidic acid
- Author
-
Shigenori Enoki, Tetsuyuki Kobayashi, Hiromu Murofushi, Mutsuko Mukai, Kimiko Murakami-Murofushi, Susumu Kobayashi, Ayako Uchiyama, Masahiro Inoue, Yuichiro Tanaka, Gabor Tigyi, Nobuyuki Kawai, Yuko Fujiwara, and Tamotsu Niki
- Subjects
RHOA ,Lung Neoplasms ,Lysophospholipids ,Phosphatidic Acids ,Antineoplastic Agents ,Biology ,Article ,Enzyme activator ,chemistry.chemical_compound ,Mice ,Cell Movement ,Cell Line, Tumor ,Lysophosphatidic acid ,Animals ,Humans ,heterocyclic compounds ,Transcellular ,Neoplasm Metastasis ,Molecular Biology ,Melanoma ,chemistry.chemical_classification ,Fatty acid ,Cell Biology ,Phosphatidic acid ,Phosphate ,Xenograft Model Antitumor Assays ,Rats ,Enzyme Activation ,chemistry ,Biochemistry ,biology.protein ,cardiovascular system ,lipids (amino acids, peptides, and proteins) ,biological phenomena, cell phenomena, and immunity ,rhoA GTP-Binding Protein - Abstract
Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.
- Published
- 2006
24. A lysophosphoinositide-specific phospholipase C distinct from other phospholipase C families in rat brain
- Author
-
Harumi Okuyama, Shiro Watanabe, T. Tsutsumi, Tetsuyuki Kobayashi, M. Miyashita, and Y. Homma
- Subjects
Gene isoform ,Immunoblotting ,Molecular Sequence Data ,Biophysics ,Cholic Acid ,Sodium Chloride ,Phosphatidylinositols ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Animals ,Phosphatidylinositol ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Antiserum ,Binding Sites ,biology ,Phospholipase C ,Hydrolysis ,Cell Membrane ,Active site ,Brain ,Cholic Acids ,Molecular biology ,Rats ,Membrane ,chemistry ,Solubility ,Type C Phospholipases ,biology.protein ,Lysophosphatidylinositol ,Lysophospholipids ,Peptides - Abstract
Distinct phospholipase C activities capable of hydrolyzing lysophosphatidylinositol (lysoPI-PLC) or phosphatidylinositol (PI-PLC) have been demonstrated in rat brain membranes. Treatment of brain membranes with 1 M NaCl or 1% sodium cholate solubilized a majority of PI-PLC activity from the membranes, whereas a significant level of lysoPI-PLC activity still remained membrane-associated. Most of the lysoPI-PLC activity was recovered in a 0.5% sodium deoxycholate-0.25 M NaCl extract which contained only low levels of PI-PLC activity. Using the separated fractions, differences between lysoPI-PLC and the known PI-PLC isoforms were examined. A specific peptide inhibitor of PI-PLC, which was previously shown to interact with active site regions common to known PI-PLC activity. Immunoblot analysis of both the lysoPI-PLC-rich and PI-PLC-rich fractions revealed that an antiserum against PI-PLC delta 1 cross-reacted with other PI-PLC isoforms, but not significantly with lysoPI-PLC. Furthermore, lysoPI-PLC was more resistant to sulfhydryl reagents than was PI-PLC. Our results indicate that lysoPI-PLC is an enzyme distinct from PI-PLC and that lysoPI-PLC possesses a different active site than known PI-PLC isoforms.
- Published
- 1995
25. The presence of Ca(2+)-independent phospholipase A1 highly specific for phosphatidylinositol in bovine brain
- Author
-
Hiroshi Ueda, Tomonari Tsutsumi, Harumi Okuyama, Masaaki Kishimoto, Shiro Watanabe, and Tetsuyuki Kobayashi
- Subjects
PLCD3 ,PLCB2 ,Biophysics ,Magnesium Chloride ,Biology ,Phospholipase ,Phosphatidylinositols ,Biochemistry ,Phospholipases A ,Substrate Specificity ,chemistry.chemical_compound ,Calcium Chloride ,Phospholipase A1 ,Phosphoinositide phospholipase C ,Animals ,Phosphatidylinositol ,Molecular Biology ,Edetic Acid ,Phospholipase A ,Phospholipase B ,Hydrolysis ,Brain ,Cell Biology ,Phospholipases A1 ,Phospholipases A2 ,chemistry ,Calcium ,Cattle - Abstract
EDTA-insensitive phospholipase A activity hydrolyzing phosphatidylinositol was detected in a bovine brain soluble fraction. This phospholipase A was purified 25-fold by sequential chromatographies of DEAE-Toyopearl, Phenyl-Toyopearl, and Ultrahydrogel 1000. The partially purified EDTA-insensitive phospholipase A showed an apparent molecular mass of 230kDa on an Ultrahydrogel 1000 column in the presence of 0.05% Triton X-100 and a pH optimum at 7.0. The enzyme was highly specific for phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine were not hydrolyzed significantly. The enzyme activity was characterized as phospholipase A 1 , and Ca 2+ and Mg 2+ were not required for its activity. These results indicate the existence of Ca 2+ -independent, phosphatidylinositol-specific metabolism besides those catalyzed by Ca 2+ -dependent phospholipase A 2 and Ca 2+ -dependent, phosphatidylinositol-specific phospholipase C.
- Published
- 1993
26. The DNA Sequence Encoding pldA Gene, the Structural Gene for Detergent-Resistant Phospholipase A of E. coli1
- Author
-
Ken Karasawa, Mutsuo Sekiguchi, Keizo Inoue, Hiroshi Mizushima, Hideo Ikeda, Nobuyoshi Chiba, Shoshichi Nojima, Hiroshi Homma, Tetsuyuki Kobayashi, and Ichiro Kudo
- Subjects
chemistry.chemical_classification ,Phospholipase A ,Structural gene ,Nucleic acid sequence ,Protein primary structure ,General Medicine ,Biology ,Biochemistry ,Molecular biology ,Amino acid ,chemistry ,Coding region ,Molecular Biology ,Gene ,Peptide sequence - Abstract
The nucleotide sequence of the pldA gene of Escherichia coli K-12, which codes for detergent-resistant phospholipase A (DR-phospholipase A), located in the outer membrane, was determined and the amino acid sequence of DR-phospholipase A was deduced. DR-phospholipase A contains 269 amino acids, resulting in a protein with a molecular weight of 30,809. It does not contain any cysteine residues and seems to be synthesized first as a precursor with a typical signal peptide composed of 20 amino acids. The NH2-terminus of the mature protein is glutamine, a polar amino acid, while other outer membrane proteins so far determined have a nonpolar amino acid there. The hydropathy profile of the deduced amino acid sequence of DR-phospholipase A was studied. Most of the region was rather hydrophilic and there were no stretches of hydrophobic amino acids. Computer analysis showed that there are no homologies between DR-phospholipase A and other extracellular phospholipases whose amino acid sequences are known. The candidates for the promoter region of the pldA gene, the 5'-flanking region, have a significantly high AT content, while the AT content of the coding region is about the same as the average AT content of the E. coli chromosome. A typical rho-independent transcription termination site is also present at the 3'-flanking region. This is the first example of the primary structure of a membrane-bound phospholipase.
- Published
- 1984
27. Purification and Characterization of Lysophospholipase Released from Rat Platelets1
- Author
-
Sayumi Higashi, Ichiro Kudo, Tetsuyuki Kobayashi, and Keizo Inoue
- Subjects
chemistry.chemical_classification ,Gel electrophoresis ,biology ,General Medicine ,Biochemistry ,Enzyme assay ,Divalent ,chemistry.chemical_compound ,Column chromatography ,Lysophosphatidylcholine ,Lysophospholipase ,chemistry ,Enzyme inhibitor ,biology.protein ,Lysophosphatidylinositol ,Molecular Biology - Abstract
Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.
- Published
- 1988
28. Nucleotide Sequence of the pldB Gene and Characteristics of Deduced Amino Acid Sequence of Lysophospholipase L2 in Escherichia coli1
- Author
-
Ichiro Kudo, Tetsuyuki Kobayashi, Keizo Inoue, Shoshichi Nojima, Hiroshi Mizushima, and Ken Karasawa
- Subjects
Signal peptide ,chemistry.chemical_classification ,Arginine ,Peripheral membrane protein ,Nucleic acid sequence ,Protein primary structure ,General Medicine ,Biology ,Biochemistry ,Amino acid ,Lysophospholipase ,chemistry ,Molecular Biology ,Peptide sequence - Abstract
The nucleotide sequence of the pldB gene of Escherichia coli K-12, which codes for lysophospholipase L2 located in the inner membrane, was determined. The deduced amino acid sequence of lysophospholipase L2 contains 340 amino acid residues, resulting in a protein with a molecular weight of 38,934. It is characterized by a high content of arginine residues (36 out of 340 residues). The amino acid sequence near the NH2-terminus of the protein is composed of a large number of polar or charged amino acid residues, suggesting that this region cannot be a signal peptide. The hydropathy profile of the deduced amino acid sequence of lysophospholipase L2 was studied. Most of the region was rather hydrophilic, and there was no stretch of hydrophobic amino acid region, such as might be predicted to traverse the lipid bilayer. These results are consistent with the experimental observation that lysophospholipase L2 is extracted by salt solution from the membrane fraction, and it may be classified as a peripheral membrane protein. Computer analysis showed that there is no homology in amino acid sequences between lysophospholipase L2 and other extracellular phospholipases, as well as detergent-resistant phospholipase A, which is another membrane-bound phospholipase in E. coli and whose DNA sequence was determined (Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inoue, K., Ideka, H., Sekiguchi, M., & Nojima, S. (1984) J. Biochem. 96, 1655-1664). This is the first report of the primary structure of a lysophospholipase.
- Published
- 1985
29. Characteristics of Detergent-Resistant Phospholipase A Overproduced in E. coli Cells Bearing Its Cloned Structural Gene1
- Author
-
Hideo Ikeda, Ichiro Kudo, Nobuyoshi Chiba, Hiroshi Homma, Tetsuyuki Kobayashi, Keizo Inoue, Mutsuo Sekiguchi, and Shoshichi Nojima
- Subjects
Phospholipase A ,Structural gene ,General Medicine ,Biology ,Phospholipase ,medicine.disease_cause ,Biochemistry ,Molecular biology ,Plasmid ,Membrane protein ,medicine ,Bacterial outer membrane ,Molecular Biology ,Gene ,Escherichia coli - Abstract
Detergent-resistant phospholipase A (DR-phospholipase A) of E. coli is a 28K-dalton protein and is exclusively located in the outer membrane. We cloned the pldA gene of E. coli, which is responsible for the activity of DR-phospholipase A. Strains bearing the plasmid which contained the pldA gene yielded a large amount of the outer membrane protein with a molecular weight of about 28K daltons and overproduced 20 to 65 times as much DR-phospholipase A activity as the wild type strain. Experiments with minicells and maxicells revealed that the pldA-containing plasmid was coding for a 28K protein. These results strongly indicated that pldA is the structural gene for DR-phospholipase A. There was apparently no difference with respect to the association of the enzyme with the envelope fraction between the overproducer and the wild type strain. The overproduced enzyme was properly transported to the outer membrane. Neither the growth rate nor the phospholipid composition of the overproducer was remarkably different from in the wild type strain. Thus, the overproduction of DR-phospholipase A apparently caused no phenotypic variations. E. coli has very excessive ability to transport and integrate the outer membrane protein.
- Published
- 1984
30. Monoclonal Antibodies against Rat Platelet Phospholipase A21
- Author
-
Ichiro Kudo, Makoto Murakami, Tetsuyuki Kobayashi, Keizo Inoue, and Masato Umeda
- Subjects
biology ,medicine.drug_class ,General Medicine ,Phospholipase ,Monoclonal antibody ,Biochemistry ,Molecular biology ,Enzyme assay ,Phospholipase A2 ,Snake venom ,Immunochemistry ,biology.protein ,medicine ,Platelet ,Antibody ,Molecular Biology - Abstract
Monoclonal antibodies which bind specifically to rat platelet phospholipase A2 have been raised. None of them bound to exocrine phospholipase A2 derived from pancreas or snake venom. All antibodies recognized the conformational structure of rat platelet phospholipase A2 supported by intramolecular disulfide bonds, since the reactivity between the antibodies and the enzyme was lost in the presence of 2-mercaptoethanol. One of them, designed MB5.2, inhibited the activity of the platelet phospholipase A2 in a dose-dependent manner. A kinetic study revealed that antibody MB5.2 apparently competed with the substrate for the active site of the enzyme. The other antibodies, designed MD7.1 and ME6.1, inhibited the binding of the enzyme to heparin. The distribution of phospholipases A2 bearing a similar determinant to rat platelet phospholipase A2 was investigated by immunoprecipitation of the enzyme activity or by an immunoblot technique. Among rat tissues, cross-reactivity was observed with phospholipases A2 from spleen, lung, and bone marrow. Extracellular phospholipase A2 detected in the peritoneal cavity of casein-treated rat was also recognized by these antibodies. Furthermore, antibody MD7.1 cross-reacted with rabbit and guinea pig platelet phospholipases A2.
- Published
- 1988
31. Isolation of Two Kinds of E. coli K-12 Mutants for Lysophospholipase L2: One with an Elevated Level of the Enzyme and the Other Defective in It1
- Author
-
Shoshichi Nojima, Ichiro Kudo, Yumiko Natori, Hiroshi Homma, Keizo Inoue, and Tetsuyuki Kobayashi
- Subjects
Phosphatidylglycerol ,chemistry.chemical_classification ,Mutant ,General Medicine ,Biology ,medicine.disease_cause ,Biochemistry ,Molecular biology ,chemistry.chemical_compound ,Enzyme ,chemistry ,Lysophospholipase ,Hydrolase ,medicine ,Inner membrane ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Escherichia coli ,Acyl group - Abstract
Two kinds of E. coli K-12 mutants for lysophospholipase L2 (located in the inner membrane) were isolated, using an improved version of the colony autoradiographic method developed by Raetz; these were, 1) strains carrying an elevated level of the enzyme and 2) strains defective or temperature-sensitive in the enzyme. Characterization of the crude lysates of these mutants revealed that the differences of lysophospholipase L2 activity are not due to the presence or absence of regulatory factors. Evidence was obtained, by using these mutants, that this lysophospholipase L2 transfers the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol, forming acyl phosphatidylglycerol.
- Published
- 1984
32. Gene Organization of pld A and pldB, the Structural Genes for Detergent-Resistant Phospholipase A and Lysophospholipase L2 of Escherichia coli1
- Author
-
Hideo Ikeda, Ichiro Kudo, Ken Karasawa, Tetsuyuki Kobayashi, Shoshichi Nojima, Keizo Inoue, and Hiroshi Homma
- Subjects
Genetics ,biology ,Structural gene ,Protein primary structure ,General Medicine ,Lambda phage ,biology.organism_classification ,medicine.disease_cause ,Biochemistry ,Molecular biology ,chemistry.chemical_compound ,Plasmid ,Lysophospholipase ,chemistry ,medicine ,Molecular Biology ,Gene ,Escherichia coli ,DNA - Abstract
The genes coding for the phospholipid degradation enzymes in E. coli, detergent-resistant (DR-) phospholipase A (pldA) and lysophospholipase L2 (pldB), were cloned together on the plasmid pKO1 (Homma, H., Kobayashi, T., Ito, Y., Kudo, I., Inoue, K., Ikeda, H., Sekiguchi, M., & Nojima, S. (1983) J. Biochem. 94, 2079-2081). To study their gene organization, a transducing lambda phage, lambda pldApldB, carrying both the pldA and pldB genes was constructed in vitro from plasmid pKO1. Viable deletion mutants of lambda pldApldB were isolated by EDTA killing, and their deleted DNA regions were determined by electron microscopic analysis of appropriate heteroduplexes. The activities of DR-phospholipase A and lysophospholipase L2 were also measured in lysates of cells infected with the deletion phages. The DNA region essential for the expression of each lipolytic activity was determined. In addition, proteins coded by the bacterial DNA on the plasmids containing the pldApldB region to various extents were detected by the maxicell system. The results showed that the product of the pldB gene is a protein with molecular weight of 40,000. It was also shown that the pldB gene is located at a region about 3 kilobase from the pldA gene.
- Published
- 1985
33. Purification and Characterization of Lysophospholipase L2 of Escherichia coli K-121
- Author
-
Ichiro Kudo, Keizo Inoue, Takao Saeki, Tetsuyuki Kobayashi, Shoshichi Nojima, and Ken Karasawa
- Subjects
Phosphatidylglycerol ,Gel electrophoresis ,Chromatofocusing ,Structural gene ,General Medicine ,Biology ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Affinity chromatography ,chemistry ,Lysophospholipase ,medicine ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Escherichia coli ,Acyl group - Abstract
Lysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene. Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and heparin-Sepharose affinity column chromatography. The final preparation showed a single protein band with a molecular weight of 38,500 daltons in SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined. It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB [Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1017-1025.] The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids. The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol. The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative. This enzyme was heat-labile and was inactivated at 55 degrees C within 5 min. The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E. coli K-12 homogenates [Doi, O. & Nojima, S. (1975) J. Biol. Chem. 250, 5208-5214].
- Published
- 1985
34. Identification and Cloning of the Gene Coding for Lysophospholipase L2 of E. coli K-121
- Author
-
Hideo Ikeda, Yuri Ito, Keizo Inoue, Hiroshi Homma, Mutsuo Sekiguchi, Shoshichi Nojima, Tetsuyuki Kobayashi, and Ichiro Kudo
- Subjects
Phospholipase A ,Chemistry ,Mutant ,Lysophospholipase L2 ,General Medicine ,Biochemistry ,Molecular biology ,enzymes and coenzymes (carbohydrates) ,Transduction (genetics) ,Plasmid ,lipids (amino acids, peptides, and proteins) ,Hybrid plasmid ,Molecular Biology ,Gene - Abstract
E. coli bearing hybrid plasmid pKOl (Oeda et al. (1981) Mol. Gen. Genet. 184, 191-199) expressed a large amount of lysophospholipase L2 activity. When a mutant which was defective in lysophospholipase L2 activity was transformed with plasmid pKOl, it overproduced lysophospholipase L2 activity. The gene responsible for the lysophospholipase L2 activity was designated as pld B. On the same hybrid plasmid another gene (pld A) coding for detergent-resistant phospholipase A (DR-phospholipase A) was also identified. These facts together with the results of a Pl transduction experiment revealed that the pld B gene must be between the pld A and met E genes on the E. coli chromosome.
- Published
- 1983
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