記述言語：英語   掲載種別：研究論文（学術雑誌）  

Gene expression profiles in synovial tissues from rheumatoid arthritis (RA) patients have yielded useful information on the pathogenetic process of the synovitis. In one group of them, sphingosine kinase 2 (SPHK2), a nuclear protein regulating cell proliferation, seemed to be highly expressed, undergoing a different pathogenetic process of synovitis. In the present study it was clarified that SPHK2 was expressed in the synovial fibroblasts of the synovial tissues obtained from the knee joints of the RA patients. In the cultured synovial fibroblasts from these patients, SPHK2 was more highly expressed than that in the human macrophage cell line, THP-1 and human dermal fibroblasts. SPHK2 was expressed in and around the nucleus and transferred to the cytoplasm and cell surface by the administration of epidermal growth factor, associated with the increased expression of sphingosine-1-phosphate. A sphingosine analogue, FTY720, which is activated by phosphorylation specifically by SPHK2, mediated apoptotic signaling of the cultured synovial fibroblasts. These findings suggest that SPHK2 may regulate the autonomous proliferation of synovial fibroblasts as one of the predisposing genes to RA and could be a target for a novel therapeutic strategy for RA.

DOI： 10.1111/j.1440-1827.2009.02381.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Innate immunity plays important roles in host defense against pathogens, but may also contribute to the development of autoimmune diseases under certain conditions. Toll-like receptors (TLRs) recognize various pathogens and induce innate immunity. We herein present a mouse model for chronic pancreatitis, which was induced by TLR3 signaling that generated the Fas/Fas ligand (FasL)-mediated cytotoxicity. An analogue of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly I:C), which is recognized by TLR3, was injected into autoimmune-prone strains: MRL/Mp mice (MRL/+), MRL/Mp mice with a deficit of Fas (MRL/lpr) and MRL/Mp mice with a deficit of functional FasL (MRL/gld). The pancreatitis in MRL/+ mice was initiated by the destruction of pancreatic ductules, and its severity was significantly higher than that in MRL/lpr mice or MRL/gld mice. Using a pancreatic duct epithelial cell line MRL/S-1 newly established from the MRL/gld mouse that lacks FasL, we showed that treatment with poly I:C significantly induced the expression of Fas on the cultured cells. MRL/S-1 cells were destructed when co-cultured with splenocytes bearing intact FasL prepared from MRL/+ or MRL/lpr mice, but the magnitude of cytotoxicity was smaller with splenocytes of MRL/gld mice. Likewise, synthetic FasL protein showed cytotoxicity on MRL/S-1 cells. Furthermore, MRL/S-1 cells expressed higher levels of chemokines after the treatment with poly I:C, suggesting that the poly I:C-mediated induction of chemokines may be responsible for recruitment of lymphoid cells to the pancreatic periductular regions. These findings indicate that TLR3 signaling generates the Fas/FasL-mediated cytotoxicity, thereby leading to the development of chronic pancreatitis.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.

DOI： 10.1016/j.cmet.2008.12.008

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掲載種別：研究論文（学術雑誌）  

Background: Apolipoprotein E allele 4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1-272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. Results: To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1-272) more strongly than intact apoE4(1-299). Further analysis showed that in Neuro-2a cells expressing apoE4(1-272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1-299). Conclusion: ApoE4(1-272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1-272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration. © 2009 Nakamura et al; licensee BioMed Central Ltd.

DOI： 10.1186/1750-1326-4-35

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掲載種別：研究論文（学術雑誌）  

Hyperlipidemia is a common feature of diabetes and is related to cardiovascular disease. The very low-density lipoprotein receptor (VLDL-R) is a member of the low-density lipoprotein receptor (LDL-R) family. It binds and internalizes triglyceride-rich lipoproteins with high specificity. We examined the etiology of hyperlipidemia in the insulin-deficient state. VLDL-R expression in heart and skeletal muscle were measured in rats with streptozotocin (STZ)-induced diabetes. STZ rats showed severe hyperlipidemia on d 21 and 28, with a dramatic decline in VLDL-R protein in skeletal muscle (>90%), heart (∼50%) and a loss of adipose tissues itself on d 28. The reduction of VLDL-R protein in skeletal muscle could not be explained simply by a decrease at the transcriptional level, because a dissociation between VLDL-R protein and mRNA expression was observed. The expression of LDL-R and LDL-R-related protein in liver showed no consistent changes. Furthermore, no effect on VLDL-triglyceride production in liver was observed in STZ rats. A decrease in postheparin plasma lipoprotein lipase activity started on d 7 and continued to d 28 at the 50% level even though severe hyperlipidemia was detected only on d 21 and 28. In rat myoblast cells, serum deprivation for 24 h induced a reduction in VLDL-R proteins. Insulin (10-6 M), but not IGF-I (10 ng/ml), restored the decreased VLDL-R proteins by serum deprivation. These results suggest that the combination of VLDL-R deficiency and reduced plasma lipoprotein lipase activity may be responsible for severe hyperlipidemia in insulin-deficient diabetes. Copyright © 2005 by The Endocrine Society.

DOI： 10.1210/en.2005-0043

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掲載種別：研究論文（学術雑誌）  

The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH2 terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized β VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease. © The Japan Society of Human Genetics and Springer-Verlag 2004.

DOI： 10.1007/s10038-004-0198-4

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掲載種別：研究論文（学術雑誌）  

Acetyl-CoA synthetase 2 (AceCS2) produces acetyl-CoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5′-flanking region of the mouse AceCS2 gene demonstrates that Krüppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.

DOI： 10.1074/jbc.M312079200

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記述言語：英語  

The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor family. In vitro and in vivo studies have shown that VLDL receptor binds triglyceride (TG)-rich lipoproteins but not LDL, and functions as a peripheral remnant lipoprotein receptor. VLDL receptor is expressed abundantly in fatty acid-active tissues (heart, skeletal muscle and fat), the brain and macrophages. It is likely that VLDL receptor functions in concert with lipoprotein lipase (LPL), which hydrolyses TG in VLDL and chylomicron. In contrast to the LDL receptor, VLDL receptor binds apolipoprotein (apo) E2/2 VLDL particles as well as apoE3/3 VLDL, and the expression is not down-regulated by intracellular lipoproteins. Recently, various functions of the VLDL receptor have been reported in lipoprotein metabolism, metabolic syndrome/atherosclerosis, cardiac fatty acid metabolism, neuronal migration and angiogenesis/tumor growth. Gene therapy of VLDL receptor into the liver showed a benefit effect for lipoprotein metabolism in both LDL receptor knockout and apoE mutant mice. Beyond its function as a peripheral lipoprotein receptor, possibilities of its physiological function have been extended to include signal transduction, angiogenesis and tumor growth.

DOI： 10.5551/jat.11.200

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.

DOI： 10.1023/A:1024184201941

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

By expression cloning using fluorescent-labeled high density lipoprotein (HDL), we isolated two clones that conferred the cell surface binding of HDL. Nucleotide sequence of the two clones revealed that one corresponds to scavenger receptor class B, type 1 (SRBI) and the other encoded a novel protein with 228 amino acids. The primary structure of the newly identified HDL-binding protein resembles GPI-anchored proteins consisting of an N-terminal signal sequence, an acidic region with a cluster of aspartate and glutamate residues, an Ly-6 motif highly conserved among the lymphocyte antigen family, and a C-terminal hydrophobic region. This newly identified HDL-binding protein designated GPI-anchored HDL-binding protein 1 (GPI-HBP1), was susceptible to phosphatidylinositol-specific phospholipase C treatment and binds HDL with high affinity (calculated K(d) = 2-3 microg/ml). Similar to SRBI, GPI-HBP1 mediates selective lipid uptake but not the protein component of HDL. Among various ligands for SRBI, HDL was most preferentially bound to GPI-HBP1. In contrast to SRBI, GPI-HBP1 lacked HDL-dependent cholesterol efflux. The GPI-HBP1 transcripts were detected with the highest levels in heart and, to a much lesser extent, in lung and liver. In situ hybridization revealed the accumulation of GPI-HBP1 transcripts in cardiac muscle cells, hepatic Kupffer cells and sinusoidal endothelium, and bronchial epithelium and alveolar macrophages in the lung.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca(2+) in response to glucose, and thereby glucose-induced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5-- islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5-- islets. Furthermore, exposure of LRP5++ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that WntLRP5 signaling contributes to the glucose-induced insulin secretion in the islets.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The VLDL (very low-density lipoprotein) receptor is a peripheral lipoprotein receptor expressing in fatty acid active tissues abundantly. In the Balb/c fasting mice, VLDL receptor as well as LPL (lipoprotein lipase), FAT (fatty acid translocase)/CD36, H-FABP (heart-type fatty acid-binding protein), ACS (acyl-CoA synthetase) and LCAD (long-chain acyl-CoA dehydrogenase) expressions increased. An electron microscopic examination indicated the lipid droplets that accumulated in the hearts of fasting Balb/c mice. During the development of SD (Sprague-Dawley) rats, VLDL receptor, LPL, FAT/CD36, H-FABP, ACS, and LCAD mRNAs concomitantly increased with growth. However, PK (pyruvate kinase) mRNA expression was negligible. In cultured neonatal rat cardiomyocytes, VLDL receptor expression increased with days in culture. Oil red-O staining showed that cardiomyocytes after 7 days in culture (when the VLDL receptor protein is present) accumulated beta-migrating VLDL. Thereby, we showed that the cardiac VLDL receptor pathway for delivery of remnant lipoprotein particles might be part of a cardiac fatty acid metabolism.

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掲載種別：研究論文（学術雑誌）  

Acyl-CoA synthetases (ACSs) play an essential role in fatty acid metabolism. ACS3 is an arachidonate-preferring enzyme expressed in a wide range of human tissues including brain, heart, placenta, prostate, skeletal muscle, testis and thymus. As an initial step to understanding the transcriptional regulation of the human ACS3 gene, we analyzed the genomic organization and transcription units of the human ACS3 gene. Sequence analysis of genomic clones demonstrates that the human ACS3 gene spans at least 80.6 kb and contains 17 exons. The human ACS3 gene was mapped between the sequence-tagged site markers D2S360 and WI-21901. Sequence inspection of the 5′-flanking region revealed potential DNA elements including CCAAT, AP-1, Oct-1, GATAs, SRY, CdxA, Nkx-2.5, c-Myb, HSF2, NF-AT, AP-2, NF-Y, and p300. A minimal promoter region required for the expression of the human ACS3 gene in melanoma G361 cells was determined. © 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(01)00714-4

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掲載種別：研究論文（学術雑誌）  

In this study, we identified and characterized two murine cDNAs encoding medium-chain acyl-CoA synthetase (MACS). One, designated MACS1, is a novel protein and the other the product of the Sa gene (Sa protein), which is preferentially expressed in spontaneously hypertensive rats. Based on the murine MACS1 sequence, we also identified the location and organization of the human MACS1 gene, showing that the human MACS1 and Sa genes are located in the opposite transcriptional direction within a 150-kilobase region on chromosome 16p13.1. Murine MACS1 and Sa protein were overexpressed in COS cells, purified to homogeneity, and characterized. Among C4-C16 fatty acids, MACS1 preferentially utilizes octanoate, whereas isobutyrate is the most preferred fatty acid among C2-C6 fatty acids for Sa protein. Like Sa gene transcript, MACS1 mRNA was detected mainly in the liver and kidney. Subcellular fractionation revealed that both MACS1 and Sa protein are localized in the mitochondrial matrix. 14C-Fatty acid incorporation studies indicated that acyl-CoAs produced by MACS1 and Sa protein are utilized mainly for oxidation.

DOI： 10.1074/jbc.M106651200

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掲載種別：研究論文（学術雑誌）  

Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5′-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (∼2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility " supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.

DOI： 10.1074/jbc.M103848200

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掲載種別：研究論文（学術雑誌）  

Using peptide sequences derived from bovine cardiac acetyl-CoA synthetase (AceCS), we isolated and characterized cDNAs for a bovine and murine cardiac enzyme designated AceCS2. We also isolated a murine cDNA encoding a hepatic type enzyme, designated AceCS1, identical to one reported recently (Luong, A., Hannah, V. C., Brown, M. S., and Goldstein, J. L. (2000) J. Biol. Chem. 275, 26458-26466). Murine AceCS1 and AceCS2 were purified to homogeneity and characterized. Among C2-C5 short and medium chain fatty acids, both enzymes preferentially utilize acetate with similar affinity. The AceCS2 transcripts are expressed in a wide range of tissues, with the highest levels in heart, and are apparently absent from the liver. The levels of AceCS2 mRNA in skeletal muscle were increased markedly under ketogenic conditions. Subcellular fractionation revealed that AceCS2 is a mitochondrial matrix enzyme. [ 14C]Acetate incorporation indicated that acetyl-CoAs produced by AceCS2 are utilized mainly for oxidation.

DOI： 10.1074/jbc.M008782200

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掲載種別：研究論文（学術雑誌）  

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring isozyme of ACS family predominantly expressed in steroidogenic tissues. Isolation and characterization of genomic clones encoding human ACS4 revealed that the genomic organization of the gene. The human ACS4 gene spans approximately 90 kb and consists of 16 exons. Sequence inspection of the 5′-flanking region revealed potential DNA elements including GATAs, p300, AP-4, SRY, CREB and MyoD. A minimal promoter region required for the expression of ACS4 in HeLa S3 cells was determined. The human ACS4 gene was mapped between the STS markers, WI-17685 and CHLC.GATA81B07 on Xq22-23 region. © 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5357

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掲載種別：研究論文（学術雑誌）  

Arachidonate released by various stimuli is rapidly reesterified into membrane phospholipids initiated by acyl-CoA synthetase (ACS) and subsequent acyl-transfer reactions. ACS4 is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. Female mice heterozygous for ACS4 deficiency become pregnant less frequently and produce small litters with extremely low transmission of the disrupted alleles. Striking morphological changes, including extremely enlarged uteri and lumina filled with numerous proliferative cysts of various sizes, were detected in ACS4+/- females. Furthermore, marked accumulation of prostaglandins was seen in the uterus of the heterozygous females. These results indicate that ACS4 modulates female fertility and uterine prostaglandin production. © 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5065

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掲載種別：研究論文（学術雑誌）  

Acyl-CoA synthetase (ACS) ligates fatty acid and CoA to produce acyl-CoA, an essential molecule in fatty acid metabolism and cell proliferation. ACS5 is a recently characterized ACS isozyme highly expressed in proliferating 3T3-L1 cells. Molecular characterization of the human ACS5 gene revealed that the gene is located on chromosome 10q25.1-q25.2, spans approximately 46 kb, comprises 21 exons and 22 introns, and encodes a 683 amino acid protein. Two major ACS5 transcripts of 2.5- and 3.7-kb are distributed in a wide range of tissues with the highest expression in uterus and spleen. Markedly increased levels of ACS5 transcripts were detected in a glioma line, A172 cells, and primary gliomas of grade IV malignancy, while ACS5 expression was found to be low in normal brain. Immunohistochemical analysis also revealed strong immunostaining with an anti-ACS5 antibody in glioblastomas. U87MG glioma cells infected with an adenovirus encoding ACS5 displayed induced cell growth on exposure to palmitate. Consistent with the induction of cell growth, the virus infected cells displayed induced uptake of palmitate. These results demonstrate a novel fatty acid-induced glioma cell growth mediated by ACS5.

DOI： 10.1038/sj.onc.1203981

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掲載種別：研究論文（学術雑誌）  

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues. We demonstrate that ACS4 expression in steroidogenic tissues in vivo is induced by adrenocorticotropic hormone (ACTH) and suppressed by glucocorticoid. ACTH also induced ACS4 protein but not its mRNA in Y1 adrenocortical tumor cells, whereas both ACS4 mRNA and protein were increased by dibutyryl cAMP (db-cAMP) and forskolin. Furthermore, the levels of ACS4 mRNA and protein in Y1 cells were induced by arachidonate. These data suggest that ACS4 expression in steroidogenic cells is regulated in coordination with induced steroidogenesis and arachidonate released by cholesterol ester hydrolase. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3207

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：SPRINGER-VERLAG TOKYO  

Short-chain acyl-CoA synthetase (SACS) is involved in the initial activation step of the metabolism of short-chain fatty acids such as acetate and propionate. Two cDNAs encoding short-chain acyl-CoA synthetases, designated SACS1 and SACS2, were isolated and characterized. The enzymes activate acetate, propionate and acetoacetate to their CoA derivatives with high efficiency. SACS1 mRNA is abundant in tissues performing active beta -oxidation such as the heart and kidney. SACS2 mRNA is present in various tissues, including the liver, and its expression is markedly increased during adipogenic differentiation from 3T3-L1 preadipocytes and embryonic fibloblasts. These data suggest that SACS1 plays a role in the oxidation of ketone bodies for energy production under ketotic conditions and SACS2 functions in the utilization of short-chain fatty acids for the biosynthesis of cellular lipid under lipogenic conditions.

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掲載種別：研究論文（学術雑誌）  

The C→U editing of RNA is widely found in plant and animal species. In mammals it is a discrete process confined to the editing of apolipoprotein B (apoB) mRNA in eutherians and the editing of the mitochondrial tRNA for glycine in marsupials. Here we have identified and characterised apoB mRNA editing in the American opossum Monodelphus domestica. The apoB mRNA editing site is highly conserved in the opossum and undergoes complete editing in the small intestine, but not in the liver or other tissues. Opossum APOBEC-1 cDNA was cloned, sequenced and expressed. The encoded protein is similar to APOBEC-1 of eutherians. Motifs previously identified as involved in zinc binding, RNA binding and catalysis, nuclear localisation and a C-terminal leucine-rich domain are all conserved. Opossum APOBEC-1 contains a seven amino acid C-terminal extension also found in humans and rabbits, but not present in rodents. The opossum APOBEC-1 gene has the same intron/exon organisation in the coding sequence as the eutherian gene. Northern blot and RT-PCR analyses and an editing assay indicate that no APOBEC-1 was expressed in the liver. Thus the far upstream promoter responsible for hepatic expression in rodents does not operate in the opossum. An APOBEC-1-like enzyme such as might be involved in C→U RNA editing of tRNA in marsupial mitochondria was not demonstrated. The activity of opossum APOBEC-1 in the presence of both chicken and rodent auxiliary editing proteins was comparable to that of other mammals. These studies extend the origins of APOBEC-1 back 170,000,000 years to marsupials and help bridge the gap in the origins of this RNA editing process between birds and eutherian mammals.

DOI： 10.1093/nar/27.13.2662

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：The Japanese Biochemical Society  

We report here the identification, characterization, and expression of a novel rat acyl-CoA synthetase (ACS) designated as ACS5. ACS5 consists of 683 amino acids and is approximately 60% identical to the previously characterized ACS1 and ACS2. ACS5 was overproduced in Escherichia coli cells and then purified to near homogeneity. The purified enzyme utilized a wide range of saturated fatty acids similar to those utilized by ACS1 and ACS5., but differed in its preference for C16-C18 unsaturated fatty acids. Northern blot analysis revealed that ACS5 mRNA is present most abundantly in the small intestine, and to a much lesser extent in the lung, liver, adrenal gland, adipose tissue, and kidney. In situ hybridization of rat ileum revealed abundant accumulation of ACS5. transcripts in foveolar epithelial cells. The hepatic level of ACS5 mRNA was significantly increased by refeeding a fat-free high sucrose diet and reduced by fasting or refeeding a high cholesterol diet, whereas that in the small intestine was not significantly altered by various dietary conditions. In contrast to the absence of ACS1 mRNA in undifferentiated 3T3-L1 preadipocytes, ACS5 mRNA was present in proliferating 3T3-L1 preadipocytes and its level remained unaltered during differentiation, suggesting that ACS5 may provide the acyl-CoA utilized for the synthesis of cellular lipids in proliferating preadipocytes.

DOI： 10.1093/oxfordjournals.jbchem.a022165

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掲載種別：研究論文（学術雑誌）  

ApoB RNA-editing enzyme (APOBEC-1) is a cytidine deaminase. Molecular modeling and mutagenesis show that APOBEC-1 is related in quaternary and tertiary structure to Escherichia coli cytidine deaminase (ECCDA). Both enzymes form a homodimer with composite active sites constructed with contributions from each monomer. Significant gaps are present in the APOBEC-1 sequence, compared to ECCDA. The combined mass of the gaps (10 kDa) matches that for the minimal RNA substrate. Their location in ECCDA suggests how ABOBEC-1 can be reshaped to accommodate an RNA substrate. In this model, the asymmetrical binding to one active site of a downstream U (equivalent to the deamination product) helps target the other active site for deamination of the upstream C substrate.

DOI： 10.1006/jmbi.1997.1506

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掲載種別：研究論文（学術雑誌）  

Genomic clones encoding the human APOBEC1 gene and its 5' flanking region have been isolated and characterized. The human gene contains five coding exons. The introns dividing these exons correspond exactly to those found in the mouse gene. The translation initiation site, ATG, is located in exon 2 at the same site as in the mouse. The 5' flanking sequence contains two Alu repeats of the Sq family. Primer extension analysis demonstrated the presence of two major transcription initiation sites. The first transcription initiation site delineates the beginning of a noncoding first exon and resides downstream of the first Alu sequence. The second transcription initiation site is within the second Alu repeat. This Alu repeat resides within the first intron, which is spliced out of the transcript from the first start site. Neither transcription initiation site has a TATA or CCAT box. Comparison with the mouse gene suggests that the Alu sequence insertion split the intestinal promoter and that subsequently the downstream Alu sequence took on a promoter function. No evidence was found for a far upstream non-tissue-specific promoter similar to that demonstrated in the mouse gene. Rather, consideration of results from the marsupial APOBEC-1 gene suggests that this upstream mouse promoter may have had a later evolutionary origin.

DOI： 10.1006/geno.1997.5110

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記述言語：英語   出版者・発行元：The Japanese Biochemical Society  

We report herein the identification of a novel member of the low-density lipoprotein receptor (LDLR) family termed LDLR-related protein 4 (LRP 4). Murine LRP 4 cDNA encodes a 1113-amino-acid type II membrane-like protein with eight ligand-binding repeats in two clusters. Southern blot analysis of genomic DNA from several different organisms suggests the presence of LRP 4 homologues in chicken lacking the gene encoding apolipoprotein E, which is recognized by the ligand-binding repeats of LDLR. LRP 4 transcripts were detected almost exclusively in heart in mouse and humans. Despite the presence of the ligand-binding repeats, COS cells transfected with LRP 4 did not show surface-binding of β-migrating very-low-density lipoprotein, suggesting that LRP 4 plays a role in a pathway other than lipoprotein metabolism.

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記述言語：英語   出版者・発行元：The Japanese Biochemical Society  

The isolation and characterization of rabbit and human cDNAs revealed a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1, 616-amino acid type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibited increased binding of apolipoprotein E (apoE)-enriched β-migrating very low density lipoprotein. Northern blotting and <i>in situ</i> hybridization revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA was increased in the liver and accumulated in cholesterol-laden foam cells of atherosclerotic lesions.

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掲載種別：研究論文（学術雑誌）  

ACS3 is a recently identified acyl-CoA synthetase (ACS) isozyme that preferentially utilizes laurate, myristate, arachidonate, and eicosapentaenoate among saturated and unsaturated long chain fatty acids. The ACS3 purified from COS cells transfected with the ACS3 cDNA was separated by SDS-PAGE into two major forms of 79 and 80 kDa. We report here that alternative translation initiation from ACS3 mRNA gives rise to these two isoforms of ACS3. In vitro mutagenesis of the ACS3 cDNA revealed that the translation of the 80-kDa and 79-kDa isoforms started from the first and second in-frame AUGs, respectively. The two isoforms of ACS3 expressed in COS cells exhibited similar levels of ACS activities toward palmitate and myristate. Immunocytochemistry of intact COS cells transfected with various ACS3 expression vectors suggested that the two forms are localized in the extranuclear compartment, where they exhibit a reticular pattern. In rat cerebrum, the 80-kDa isoform of ACS3 was detected mainly in the microsomal fraction. Only a trace amount of the 79-kDa isoform was detected in rat cerebrum, whereas both forms were detected in rat glioma cell line KEG1 cells.

DOI： 10.1093/oxfordjournals.jbchem.a021731

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1006/geno.1997.4710

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掲載種別：研究論文（学術雑誌）  

We report herein the cDNA cloning of a novel rat acyl-CoA synthetase (ACS) that preferentially uses arachidonate and eicosapentaenoate. This newly identified ACS (designated ACS4) contains 670 amino acids and is 68% identical to rat ACS3, a previously characterized ACS that is highly expressed in brain. ACS4 was overproduced in Escherichia coli and the resulting enzyme was purified to homogeneity. The purified enzyme utilizes arachidonate and eicosapentaenoate most preferentially among C8-C22 saturated fatty acids and C14-C22 unsaturated fatty acids. Kinetic analyses revealed that the enzyme has a high affinity for arachidonate and eicosapentaenoate and low affinity for palmitate. ACS4 transcripts are detectable in a wide range of tissues, with the highest level in adrenal gland. Immunoreactivity to ACS4 was detected in the zona fasciculata and reticularis of adrenal gland, in the corpus luteum and stromal luteinized cells in ovary, and in the Leydig cells of testis.

DOI： 10.1073/pnas.94.7.2880

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掲載種別：研究論文（学術雑誌）  

Two types of acyl-CoA synthetase (ACS), designated ACS1 and ACS2, are structurally similar isozymes with different tissue distributions. The two enzymes are organized into the following five regions: an NH2 terminus; two luciferase-like regions; a linker connecting the luciferase-like regions; a COOH terminus. Under the control of a lac promoter, rat ACS1 and ACS2 were overproduced in Escherichia coli and purified to homogeneity. The specific activities of the purified ACS1 and ACS2 were 26.2 μmol · min-1 · mg-1 and 7.4 μmol · min-1 · mg-1, respectively, and the most efficiently utilized saturated fatty acids were those with 10-18 carbon atoms. Among unsaturated fatty acids with 16-22 carbon atoms, the most preferred substrates were palmitoleate, oleate and linoleate for ACS1, and, for ACS2, oleate, arachidonate, eicosapentaenoate and docosahexaenoate. To determine the functionally important regions in the ACS isozymes, we constructed five ACS1 mutants lacking each of the five regions. Introduction of these mutants into E. coli revealed that all five regions in ACS1 are required for functional expression of the enzyme in E. coli; deletion of any one of the five regions almost completely abolished the enzyme activity.

DOI： 10.1111/j.1432-1033.1996.0186r.x

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掲載種別：研究論文（学術雑誌）  

Isolation and characterization of a rat brain cDNA identified a third acyl-CoA synthetase (ACS) designated ACS3. The deduced amino acid sequence of the cDNA revealed that ACS3 consists of 720 amino acids and exhibits a structural architecture common to ACSs from various origins. ACS3 expressed in COS cells was purified to near homogeneity. The purified ACS3 resolved by SDS-polyacrylamide gel electrophoresis into two major proteins of 79 and 80 kDa. Cell-free translation of a synthetic mRNA encoding the entire region of ACS3 revealed that the two isoforms were derived from the same mRNA. The purified ACS3 utilizes laurate and myristate most efficiently among C8- C22 saturated fatty acids and arachidonate and eicosapentaenoate among C16-C20 unsaturated fatty acids. Northern blot analysis revealed that ACS3 mRNA is most abundant in brain and, to a much lesser extent, in lung, adrenal gland, kidney, and small intestine. During the development of the rat brain, expression of ACS3 mRNA reached a maximum level at 15 days after birth and then declined gradually to 10% of the maximum in the adult brain.

DOI： 10.1074/jbc.271.28.16748

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掲載種別：研究論文（学術雑誌）  

Nucleotide sequence analysis of six independently isolated cDNAs for rat acyl-CoA synthetase (ACS) revealed three forms of ACS mRNA, designated form-A, -B, and -C mRNAs, which differ in their 5′-untranslated regions. Form-A mRNA was preferentially detected in normal and peroxisome-induced livers, whereas form-B mRNA was found in peroxisome-induced livers but not in normal livers and hearts, and form-C mRNA was preferentially found in normal hearts and peroxisome-induced livers. Analysis of two overlapping genomic clones for the rat ACS gene revealed that the three 5′-untranslated regions of the mRNAs are individually encoded by three different exons located within a 20-kilobase genomic fragment. The transcription start sites of the three forms of ACS mRNA were determined and nucleotide sequences of 5′-upstream regions of the three 5′-end exons were determined. The 5′-upstream regions were fused to the chloramphenicol acetyltransferase gene and transcription units of the three forms of ACS mRNAs were determined. These data indicate that the three forms of ACS mRNA with 5′-end heterogeneity are generated by alternative transcription from three promoters in the rat ACS gene.

DOI： 10.1074/jbc.270.16.9676

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掲載種別：研究論文（学術雑誌）  

The site-specific C to U editing of apolipoprotein B100 (apoB100) mRNA requires a 27 kDa protein (p27) with homology to cytidine deaminase. Here, we show that p27 is a zinc-containing deaminase, which operates catalytically like the E. coli enzyme that acts on monomeric substrate. In contrast with the bacterial enzyme that does not bind RNA, p27 interacts with its polymeric apoB m RNA substrate at AU sequences adjacent to the editing site. This interaction is necessary for editing. RNA binding is mediated through amino acid residues involved in zinc coordination, in proton shuttling, and in forming the αβα structure that encompasses the active site. However, certain mutations that inactivate the enzyme do not affect RNA binding. Thus, RNA binding does not require a catalytically active site. The acquisition of polymeric substrate binding provides a route for the evolution of this editing enzyme from one that acts on monomeric substrates. © 1995.

DOI： 10.1016/0092-8674(95)90328-3

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掲載種別：研究論文（学術雑誌）  

Acetyl-CoA synthetase from bovine heart has been purified to homogeneity and been crystallized. The purification procedure involves ammonium sulfate precipitation and subsequent column chromatography on DEAE-Sepharose, Blue-Sepharose, CoA-Agarose and Superose 6. The purified enzyme has a specific activity of 45 units/mg protein, and its molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis is approximately 72, 000. The purified enzyme specifically utilizes acetate, ATP and CoA. Apparent Km values of the purified enzyme for acetate, CoA, and ATP were 0.16 mM, 0.14 mM and 0.25 mM, respectively. Limited digestion with trypsin, subtilisin BPN' and chymotrypsin revealed that the enzyme contains a 56 k segment resistant to these proteases. Secondary structure contents of the purified enzyme and the 56 k Cryptic fragment were analyzed by circular dichroism measurement. The intact molecule contains 30% α-helix and 30% β-structure, and trypsin digests α-helix rich regions more substantially. Western blot analysis of rat tissue homogenates by specific antibodies against the purified enzyme indicated that the 72 k enzyme is present in a wide variety of tissues and is most abundant in heart and kidney. © 1995, Tohoku University Medical Press. All rights reserved.

DOI： 10.1620/tjem.175.55

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：The Japanese Biochemical Society  

Complementary DNA clones encoding a protein highly homologous to the previously characterized long-chain acyl-CoA synthetase (LACS) in liver were isolated from rat brain cDNA libraries. This protein consists of 697 amino acids and has 64.7% identity with the rat liver LACS sequence. The brain protein and the liver LACS share essentially the same domain structure, having two regions similar to those of click beetle luciferase and a long discrete gap flanking the similar domains. A significant sequence similarity was found between the brain protein and malaria octapeptide-repeat antigen, suggesting a functional similarity. COS cells transfected with the cDNA for the brain protein expressed LACS activity with slightly different fatty acid specificity from that of the liver LACS. This new LACS is expressed predominantly in brain and, to a much lesser extent, in heart and adrenal. The 2.9- and 6.3-kb mRNAs coding for the brain enzyme are coregulated with the development of brain, suggesting the physiological importance of the enzyme in fatty acid metabolism in brain. © 1992 BY The Journal of Biochemistry.

DOI： 10.1093/oxfordjournals.jbchem.a123737

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：The Japanese Biochemical Society  

A complementary DNA clone encoding the entire human long-chain acyl-CoA synthetase was isolated and the total 698-amino acid sequence was deduced. The amino acid sequence of human long-chain acyl-CoA synthetase shows 84.9% identity to that of rat long-chain acyl-CoA synthetase. The nucleotide sequences of the protein coding regions between human and rat long-chain acyl-CoA synthetase mRNAs are highly conserved (85.6%), whereas those of the 3′ untranslated regions are less conserved (72%). The location of the human long-chain acyl-CoA synthetase gene was identified on chromosome 4 by spot hybridization of flow-sorted chromosomes. Computer-assisted homology search revealed a significant similarity of the enzyme with the enzymes of the luciferase family. Based on this similarity, the structure of human long-chain acyl-CoA synthetase can be divided into five domains: the N-terminus, two domains similar to those in enzymes of the luciferase family, a long gap region between the similar domains and the C-terminus. © 1992 BY The Journal of Biochemistry.

DOI： 10.1093/oxfordjournals.jbchem.a123707

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記述言語：英語   出版者・発行元：(一社)日本解剖学会  

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記述言語：日本語   出版者・発行元：(一社)日本病理学会  

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記述言語：日本語   出版者・発行元：(一社)日本動脈硬化学会  

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記述言語：日本語   出版者・発行元：(株)羊土社  

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記述言語：英語   出版者・発行元：日本神経化学会  

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記述言語：日本語   出版者・発行元：ライフサイエンス出版(株)  

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記述言語：日本語   出版者・発行元：(一社)日本動脈硬化学会  

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記述言語：英語   出版者・発行元：(一社)日本動脈硬化学会  

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記述言語：英語   出版者・発行元：(一社)日本動脈硬化学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER SCI IRELAND LTD  

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記述言語：日本語   出版者・発行元：(一社)日本動脈硬化学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：日本語   出版者・発行元：(一社)日本動脈硬化学会  

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記述言語：日本語   出版者・発行元：(一社)日本内分泌学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：医歯薬出版(株)  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：日本脂質生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：日本語   出版者・発行元：日本脂質生化学会  

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