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Find video protocols related to scientific articles indexed in Pubmed.
A case of nonfunctioning pancreatic endocrine tumor with atypical imaging findings due to prominent fibrosis of the tumor stroma.
J Nippon Med Sch
PUBLISHED: 07-08-2014
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The patient, a 56-year-old woman, was found during routine checkup to have a disorder of hepatic function. Abdominal ultrasonography showed an ill-defined hypoechoic mass in the head and body of the pancreas; however, no blood-flow signal was observed within the tumor on Doppler ultrasonography. Abdominal computed tomography showed a low-density area in the arterial and portal venous phases. The lesion was visualized as an area of low signal intensity on both T1- and T2-weighted magnetic resonance images, whereas fluorodeoxyglucose positron emission tomography showed fluorodeoxyglucose accumulation in the tumor. Although a preoperative diagnosis was difficult to make, a rapid cytologic examination revealed evidence of a pancreatic endocrine tumor, and subtotal stomach-preserving pancreaticoduodenectomy with portal vein resection was performed. Histopathological examination showed tumor cell nests scattered in abundant fibrotic tissue; the tumor cells had proliferated in a cord-like fashion and showed immunostaining for chromogranin A. Staining for fibroblast activation protein ? was seen in the fibroblastic cells contained within the fibrous stroma surrounding the tumor cell nests, whereas both the fibroblastic cells in the tumor and those in the stroma showed a high rate of staining for thrombospondin. We presume that tumor-associated fibroblasts were involved in the fibrosis of the tumor stroma.
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Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2.
J. Lipid Res.
PUBLISHED: 05-21-2014
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Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.
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12-Hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor.
J. Exp. Med.
PUBLISHED: 05-12-2014
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Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G protein-coupled receptor (GPCR) for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. As mouse BLT2 is highly expressed in epidermal keratinocytes, we investigated the role of the 12-HHT/BLT2 axis in skin wound healing processes. 12-HHT accumulated in the wound fluid in mice, and BLT2-deficient mice exhibited impaired re-epithelialization and delayed wound closure after skin punching. Aspirin administration reduced 12-HHT production and resulted in delayed wound closure in wild-type mice, which was abrogated in BLT2-deficient mice. In vitro scratch assay using primary keratinocytes and a keratinocyte cell line also showed that the 12-HHT/BLT2 axis accelerated wound closure through the production of tumor necrosis factor ? (TNF) and matrix metalloproteinases (MMPs). A synthetic BLT2 agonist accelerated wound closure in cultured cells as well as in C57BL/6J and diabetic mice. These results identify a novel mechanism underlying the action of the 12-HHT/BLT2 axis in epidermal keratinocytes and accordingly suggest the use of BLT2 agonists as therapeutic agents to accelerate wound healing, particularly for intractable wounds, such as diabetic ulcers.
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Lysophospholipid acyltransferases mediate phosphatidylcholine diversification to achieve the physical properties required in vivo.
Cell Metab.
PUBLISHED: 04-18-2014
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The acyl-chain composition of the major mammalian phospholipid phosphatidylcholine (PC) is distinct in various tissues. Although it was classically suggested that PC diversity is acquired through acyl-chain remodeling, the mechanisms and biological relevance of acyl-chain diversity remain unclear. Here, we show that differences in the substrate selectivity of lysophospholipid acyltransferases regulate tissue PC acyl-chain composition through contribution of both the de novo and remodeling pathways, depending on the fatty acid species. Unexpectedly, while dipalmitoyl-PC (DPPC) is enriched through the remodeling pathway, several polyunsaturated PC molecules accumulate during the de novo pathway. We confirmed this concept for DPPC in pulmonary surfactant and showed that the biophysical properties of this lipid are important to prevent the early onset of acute lung injury. We propose a model of harmonized processes for phospholipid diversification to satisfy in vivo requirements, with an example of its biological relevance.
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Rapid production of platelet-activating factor is induced by protein kinase C?-mediated phosphorylation of lysophosphatidylcholine acyltransferase 2 protein.
J. Biol. Chem.
PUBLISHED: 04-17-2014
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Platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is synthesized rapidly in response to extracellular stimuli by the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). We have reported previously that lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: rapid activation (30 s) after PAF stimulation and minutes to hours after LPS stimulation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) was later identified as a Ca(2+)-dependent lyso-PAFAT. However, the mechanism of rapid lyso-PAFAT activation within 30 s has not been elucidated. Here we show a new signaling pathway for rapid biosynthesis of PAF that is mediated by phosphorylation of LPCAT2 at Ser-34. Stimulation by either PAF or ATP resulted in PKC?-mediated phosphorylation of LPCAT2 to enhance lyso-PAFAT activity and rapid PAF production. Biochemical analyses showed that the phosphorylation of Ser-34 resulted in augmentation of Vmax with minimal Km change. Our results offer an answer for the previously unknown mechanism of rapid PAF production.
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Interplay between CXCR2 and BLT1 facilitates neutrophil infiltration and resultant keratinocyte activation in a murine model of imiquimod-induced psoriasis.
J. Immunol.
PUBLISHED: 03-24-2014
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Psoriasis is an inflammatory skin disease with accelerated epidermal cell turnover. Neutrophil accumulation in the skin is one of the histological characteristics of psoriasis. However, the precise mechanism and role of neutrophil infiltration remain largely unknown. In this article, we show that orchestrated action of CXCR2 and leukotriene B4 receptor BLT1 plays a key role in neutrophil recruitment during the development of imiquimod (IMQ)-induced psoriatic skin lesions in mice. Depletion of neutrophils with anti-Ly-6G Ab ameliorated the disease severity, along with reduced expression of proinflammatory cytokine IL-1? in the skin. Furthermore, CXCR2 and BLT1 coordinately promote neutrophil infiltration into the skin during the early phase of IMQ-induced inflammation. In vitro, CXCR2 ligands augment leukotriene B4 production by murine neutrophils, which, in turn, amplifies chemokine-mediated neutrophil chemotaxis via BLT1 in autocrine and/or paracrine manners. In agreement with the increased IL-19 expression in IMQ-treated mouse skin, IL-1? markedly upregulated expression of acanthosis-inducing cytokine IL-19 in human keratinocytes. We propose that coordination of chemokines, lipids, and cytokines with multiple positive feedback loops might drive the pathogenesis of psoriasis and, possibly, other inflammatory diseases as well. Interference to this positive feedback or its downstream effectors could be targets of novel anti-inflammatory treatment.
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Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells.
J. Lipid Res.
PUBLISHED: 03-19-2014
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Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained.
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Fever screening of seasonal influenza patients using a cost-effective thermopile array with small pixels for close-range thermometry.
Int. J. Infect. Dis.
PUBLISHED: 02-07-2014
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Infrared thermography systems have been used for fever screening at many airports since the outbreak of severe acute respiratory syndrome (SARS) in 2003. However, many of these systems are expensive and non-portable. Therefore, we developed a cost-effective and compact (2.9 × 5.8 × 2.0 cm) thermopile array for fever screening of patients with infectious diseases in the clinical setting.
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Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7.
Br. J. Pharmacol.
PUBLISHED: 02-06-2014
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The endogenous ligands for the LT, lipoxin (LX) and oxoeicosanoid receptors are bioactive products produced by the action of the lipoxygenase family of enzymes. The LT receptors BLT1 and BLT2 , are activated by LTB4 and the CysLT1 and CysLT2 receptors are activated by the cysteinyl-LTs, whereas oxoeicosanoids exert their action through the OXE receptor. In contrast to these pro-inflammatory mediators, LXA4 transduces responses associated with the resolution of inflammation through the receptor FPR2/ALX (ALX/FPR2). The aim of the present review is to give a state of the field on these receptors, with focus on recent important findings. For example, BLT1 receptor signalling in cancer and the dual role of the BLT2 receptor in pro- and anti-inflammatory actions have added more complexity to lipid mediator signalling. Furthermore, a cross-talk between the CysLT and P2Y receptor systems has been described, and also the presence of novel receptors for cysteinyl-LTs, such as GPR17 and GPR99. Finally, lipoxygenase metabolites derived from ?-3 essential polyunsaturated acids, the resolvins, activate the receptors GPR32 and ChemR23. In conclusion, the receptors for the lipoxygenase products make up a sophisticated and tightly controlled system of endogenous pro- and anti-inflammatory signalling in physiology and pathology.
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Predominant role of cytosolic phospholipase A2? in dioxin-induced neonatal hydronephrosis in mice.
Sci Rep
PUBLISHED: 01-24-2014
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Hydronephrosis is a common disease characterized by dilation of the renal pelvis and calices, resulting in loss of kidney function in the most severe cases. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces nonobstructive hydronephrosis in mouse neonates through upregulation of prostaglandin E2 (PGE2) synthesis pathway consisting of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) by a yet unknown mechanism. We here studied possible involvement of cytosolic phospholipase A2? (cPLA2?) in this mechanism. To this end, we used a cPLA2?-null mouse model and found that cPLA2? has a significant role in the upregulation of the PGE2 synthesis pathway through a noncanonical pathway of aryl hydrocarbon receptor. This study is the first to demonstrate the predominant role of cPLA2? in hydronephrosis. Elucidation of the pathway leading to the onset of hydronephrosis using the TCDD-exposed mouse model will deepen our understanding of the molecular basis of nonobstructive hydronephrosis in humans.
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A novel lysophosphatidic acid acyltransferase enzyme (LPAAT4) with a possible role for incorporating docosahexaenoic acid into brain glycerophospholipids.
Biochem. Biophys. Res. Commun.
PUBLISHED: 12-03-2013
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Glycerophospholipids are important components of cellular membranes, required for constructing structural barriers, and for providing precursors of bioactive lipid mediators. Lysophosphatidic acid acyltransferases (LPAATs) are enzymes known to function in the de novo glycerophospholipid biosynthetic pathway (Kennedy pathway), using lysophosphatidic acid (LPA) and acyl-CoA to form phosphatidic acid (PA). Until now, three LPAATs (LPAAT1, 2, and 3) have been reported from the 1-acyl-glycerol-3-phosphate O-acyltransferase (AGPAT) family. In this study, we identified a fourth LPAAT enzyme, LPAAT4, previously known as an uncharacterized enzyme AGPAT4 (LPAAT?), from the AGPAT family. Although LPAAT4 was known to contain AGPAT motifs essential for acyltransferase activities, detailed biochemical properties were unknown. Here, we found that mouse LPAAT4 (mLPAAT4) possesses LPAAT activity with high acyl-CoA specificity for polyunsaturated fatty acyl-CoA, especially docosahexaenoyl-CoA (22:6-CoA, DHA-CoA). mLPAAT4 was distributed in many tissues, with relatively high expression in the brain, rich in docosahexaenoic acid (DHA, 22:6). mLPAAT4 siRNA in a neuronal cell line, Neuro 2A, caused a decrease in LPAAT activity with 22:6-CoA, suggesting that mLPAAT4 functions endogenously. siRNA in Neuro 2A cells caused a decrease in 18:0-22:6 PC, whereas mLPAAT4 overexpression in Chinese hamster ovary (CHO)-K1 cells caused an increase in this species. Although DHA is considered to have many important functions for the brain, the mechanism of its incorporation into glycerophospholipids is unknown. LPAAT4 might have a significant role for maintaining DHA in neural membranes. Identification of LPAAT4 will possibly contribute to understanding the regulation and the biological roles of DHA-containing glycerophospholipids in the brain.
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TDAG8 activation inhibits osteoclastic bone resorption.
FASEB J.
PUBLISHED: 11-12-2013
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Although the roles of acids in bone metabolism are well characterized, the function of proton-sensing receptors in bone metabolism remains to be explored. In this study, we evaluated the role of proton-sensing receptor T-cell death-associated gene 8 (TDAG8) in osteoclastic activity during bone loss after ovariectomy. Through observations of bone mineral content, we found that pathological bone resorption was significantly exacerbated in mice homozygous for a gene trap mutation in the Tdag8 gene. Furthermore, osteoclasts from the homozygous mutant mice resorbed calcium in vitro more than the osteoclasts from the heterozygous mice did. Impaired osteoclast formation under acidic conditions was ameliorated in cultures of bone marrow cells by Tdag8 gene mutation. Extracellular acidification changed the cell morphology of osteoclasts via the TDAG8-Rho signaling pathway. These results suggest that the enhancement of TDAG8 function represents a new strategy for preventing bone resorption diseases, such as osteoporosis.-Hikiji, H., Endo, D., Horie, K., Harayama, T., Akahoshi, N., Igarashi, H., Kihara, Y., Yanagida, K., Takeda, J., Koji, T., Shimizu, T., Satoshi, I. TDAG8 activation inhibits osteoclastic bone resorption.
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Antiobese function of platelet-activating factor: increased adiposity in platelet-activating factor receptor-deficient mice with age.
FASEB J.
PUBLISHED: 10-07-2013
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Platelet-activating factor receptor (PAFR)-deficient mice developed a more severe obese state characterized by higher body mass (?25%) and epididymal fat mass (?55%) with age than that of wild-type (WT) littermates. PAFR-deficient mice did not show changes in the expression of critical genes involved in anabolic and catabolic metabolism in adipose, liver, and muscle tissues between 6 and 36 wk. However, a 38-81% reduction in ?3/?1-adrenergic receptor (AR) and uncoupling protein 1 (UCP1) mRNA and protein levels was observed in the interscapular brown adipose tissue (BAT) of PAFR-deficient mice. Whereas a single injection of the ?3-adrenergic agonist, CL-316,243 (25 ?g/kg) increased temperatures in the brown fat and rectums of WT mice, this increase in temperature was markedly suppressed in PAFR-deficient mice. Acetyl-CoA:lyso-platelet-activating factor (PAF) acetyltransferase, which is involved in PAF biosynthesis, and the PAF receptor were predominantly localized in BAT macrophages, whereas brown adipocytes possessed the enzyme and functional PAF receptors. The stimulation of brown adipocytes by PAF induced the expression of ?3-AR mRNA and protein (1.5- and 1.9-fold, respectively), but not that of UCP1. These results indicate that obesity in PAFR-deficient mice resulted from impaired BAT activity and suggest that the antiobese function of PAF occurs through ?3-AR/UCP1 expression in BAT.-Sugatani, J., Sadamitsu, S., Yamaguchi, M., Yamazaki, Y., Higa, R., Hattori, Y., Uchida, T., Ikari, A., Sugiyama, W., Watanabe, T., Ishii, S., Miwa, M., Shimizu, T. Antiobese function of platelet-activating factor: increased adiposity in platelet-activating factor receptor-deficient mice with age.
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Identification of Sec14-like 3 as a novel lipid-packing sensor in the lung.
FASEB J.
PUBLISHED: 09-09-2013
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Pulmonary surfactant, a complex composed primarily of lipids and associated proteins, is synthesized in alveolar type II (ATII) cells and secreted into alveoli to prevent collapse during respiration. Although numerous studies have clarified the fundamental roles of pulmonary surfactant, the molecular mechanisms of transport and secretion of pulmonary surfactant remain totally unknown. Thus, we screened candidate genes by comparing genes with the expressed sequence tag (EST) libraries of embryonic and adult lungs by using the digital differential display method in the National Center for Biotechnology Information (NCBI) database. We identified Sec14-like 3 (Sec14L3) as a new class of lipid-associated proteins highly expressed in ATII cells. We found that Sec14L3 expression is >100-fold increased during the perinatal period in the lung. Furthermore, Sec14L3 bound to small-sized liposomes (30 nm in diameter), but not to large-sized liposomes (100 nm diameter), through its Sec14 domain. Because of the increased curvature, lipid-packing defects are more likely to occur in small-sized liposomes than in large-sized liposomes. Based on these results, we conclude that Sec14L3 is a new class of lipid-packing sensor. Sec14L3 may play important roles in the lung, such as intracellular lipid transport, surfactant maturation, and endo/exocytosis.-Hishikawa, D., Shindou, H., Harayama, T., Ogasawara, R., Suwabe, A., Shimizu, T. Identification of Sec14-like 3 as a novel lipid-packing sensor in the lung.
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Acute inhibition of diacylglycerol lipase blocks endocannabinoid-mediated retrograde signalling: evidence for on-demand biosynthesis of 2-arachidonoylglycerol.
J. Physiol. (Lond.)
PUBLISHED: 07-15-2013
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The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase ? (DGL?) is one of the best-characterized retrograde messengers at central synapses. It has been thought that 2-AG is produced on demand upon activation of postsynaptic neurons. However, recent studies propose that 2-AG is pre-synthesized by DGL? and stored in neurons, and that 2-AG is released from such pre-formed pools without the participation of DGL?. To address whether the 2-AG source for retrograde signalling is the on-demand biosynthesis by DGL? or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca(2+) elevation, Gq/11 protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca(2+)-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre-Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca(2+) elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGL? rather than mobilization from presumptive pre-formed pools.
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Alkylglycerol monooxygenase as a potential modulator for PAF synthesis in macrophages.
Biochem. Biophys. Res. Commun.
PUBLISHED: 05-21-2013
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Alkylglycerol monooxygenase (AGMO, glyceryl ether monooxygenase) is an enzyme known to catalyze the cleavage of the O-alkyl bond of glyceryl ether lipids. Identification of the gene encoding AGMO was reported recently, however, the involvement of AGMO in modulating cellular lipids has not been reported until now. In this report, we investigate a possible role for AGMO in macrophage platelet-activating factor (PAF) production. AGMO mRNA expression levels decreased with lipopolysaccharide (LPS) treatments in mouse peritoneal macrophages and RAW264.7 cells. Tetrahydrobiopterin-dependent conversion of lyso-PAF to glycerophosphocholine in the microsomal fraction was also reduced in LPS-treated RAW264.7 cells. In the LPS-treated cells, both lyso-PAF and PAF levels increased. Moreover, exogenously expressed AGMO caused a reduction in cellular lyso-PAF and PAF levels in HEK293 cells. Collectively, our results suggest a possible mechanism for AGMO in modulating macrophage PAF production by regulating cellular lyso-PAF levels.
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Generation of membrane diversity by lysophospholipid acyltransferases.
J. Biochem.
PUBLISHED: 05-21-2013
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Glycerophospholipids are main components of cellular membranes and have numerous structural and functional roles to regulate cellular functions. Polyunsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, are mainly located at the sn-2, but not the sn-1 position of glycerophospholipids in an asymmetrical manner and the fatty acid compositions at both the sn-1 and sn-2 positions differ in various cell types and tissues. Asymmetry and diversity of membrane glycerophospholipids are generated in the remodelling pathway (Lands cycle), which are conducted by the concerted actions of phospholipases A2 (PLA2s) and lysophospholipid acyltransferases (LPLATs). The Lands cycle was first reported in the 1950s. While PLA2s have been well characterized, little is known about the LPLATs. Recently, several laboratories, including ours, isolated LPLATs that function in the Lands cycle from the 1-acylglycerol-3-phosphate O-acyltransferase family and the membrane bound O-acyltransferases family. In this review, we summarize recent studies on cloning and characterization of LPLATs that contribute to membrane asymmetry and diversity.
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Leukotriene B4 type-1 receptor signaling promotes liver repair after hepatic ischemia/reperfusion injury through the enhancement of macrophage recruitment.
FASEB J.
PUBLISHED: 04-29-2013
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Recruited macrophages play a critical role in liver repair after acute liver injury. Leukotriene B4 (LTB4) is a potent chemoattractant for macrophages. In this study, we investigated the role of LTB4 receptor type 1 (BLT1) in liver repair during hepatic ischemia/reperfusion (I/R) injury. BLT1-knockout mice (BLT1(-/-)) or their wild-type counterparts (WT) were subjected to partial hepatic I/R. Compared with WT, BLT1(-/-) exhibited delayed liver repair and hepatocyte proliferation accompanied by a 70% reduction in the recruitment of macrophages and a 70-80% attenuation in hepatic expression of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1). Disruption of BLT1 signaling also reduced the expression of EGF by 67% on recruited macrophages expressing VEGFR1 in the injured liver. Treatment of WT mice with an EGF-neutralizing antibody delayed liver repair and reduced macrophage recruitment, compared with control immunoglobulin G (IgG). BLT1 signaling enhanced the expression of VEGF, VEGFR1, and EGF in isolated peritoneal macrophages in vitro. These results indicate that BLT1 signaling plays a role in liver repair after hepatic I/R through enhanced expression of EGF in recruited macrophages and that the development of a specific agonist for BLT1 could be useful for liver recovery from acute liver injury.
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Platelet-activating factor receptor knockout mice are protected from MPTP-induced dopaminergic degeneration.
Neurochem. Int.
PUBLISHED: 04-21-2013
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Platelet-activating factor (PAF), a potent mediator of inflammatory and immune responses, plays various roles in neuronal functions. However, little is known about the role of PAF/platelet-activating factor receptor (PAF-R) in Parkinsons disease. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in significant increases in PAF species in the striatum of wild-type mice. These increases paralleled PAF-R gene expression in wild-type mice. Although nuclear factor kappa B (NF-?B) DNA-binding activity was increased significantly in MPTP-treated wild-type mice, this increase was not significant in PAF-R antagonist ginkgolide B (GB)-treated mice or PAF-R knockout (PAF-R(-/-)) mice. Pyrrolidine dithiocarbamate (PDTC), an NF-?B inhibitor, significantly ameliorated the dopaminergic deficits induced by MPTP in wild-type mice. MPTP treatment significantly increased oxidative damage, the immunoreactivity of ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglial cells, and microglial differentiation of the M1 type in the striatum of wild-type mice. Consistently, PDTC significantly attenuated MPTP-induced behavioral impairments in wild-type mice. However, dopaminergic deficits, oxidative damage, reactive microglial cells, and behavioral impairments induced by MPTP were not significantly observed in GB-treated mice or PAF-R(-/-) mice. PDTC did not significantly alter the attenuations evident in MPTP-treated PAF-R(-/-) mice, indicating that NF-?B is a critical target for neurotoxic modulation of PAF-R. We propose for the first time that PAF/PAF-R can mediate dopaminergic degeneration via an NF-?B-dependent signaling process.
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Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.
Nat. Immunol.
PUBLISHED: 03-11-2013
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Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.
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Comparative study of phase transitions in BaTiO3 thin films grown on (001)- and (110)-oriented SrTiO3 substrate.
J Phys Condens Matter
PUBLISHED: 02-19-2013
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An increase of the Curie temperature, TC, was demonstrated for BaTiO3 (BTO) epitaxial thin films grown by a pulsed laser deposition technique on (001) and (110) SrTiO3 (STO) substrates. The T(C) values for the BTO films on (001) and (110) STO were determined, from the temperature dependence of the lattice constants, to be 720 and 550 K, respectively, both of which are higher than that for bulk BTO. The increase of T(C) for the film on (001) BTO is suggested to stem from a large tetragonal deformation with a tetragonality of ~1.02 due to a two-dimensional clamping effect, which persists even in the paraelectric phase. That for the (110) BTO film, on the other hand, could be caused by a one-dimensional clamping effect due to uniaxial strain.
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Excitotoxicity-induced immediate surge in hippocampal prostanoid production has latent effects that promote chronic progressive neuronal death.
Prostaglandins Leukot. Essent. Fatty Acids
PUBLISHED: 02-06-2013
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Excitotoxicity is involved in neurodegenerative conditions. We investigated the pathological significance of a surge in prostaglandin production immediately after kainic acid (KA) administration [initial phase], followed by a sustained moderate elevation in prostaglandin level [late phase] in the hippocampus of juvenile rats. Numerous pyknotic hippocampal neurons were observed 72 h after KA treatment; this number remained elevated on days 10 and 30. Gross hippocampal atrophy was observed on days 10 and 30. Pre-treatment with indomethacin ameliorated neuronal death on days 10 and 30, and prevented hippocampal atrophy on day 30. Microglial response was moderated by the indomethacin pre-treatment. Blockade of only late-phase prostaglandin production by post-treatment with indomethacin ameliorated neuronal death on day 30. These findings suggest a role for initial-phase prostaglandin production in chronic progressive neuronal death, which is exacerbated by late-phase prostaglandin production. Blockade of prostaglandin production has therapeutic implications in preventing long-term neurological sequelae following excitotoxic brain damage.
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Amino acid residues of G-protein-coupled receptors critical for endoplasmic reticulum export and trafficking.
Meth. Enzymol.
PUBLISHED: 01-29-2013
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Analysis of the structural features of rhodopsin-type G-protein-coupled receptors (GPCRs) revealed the existence of an additional ?-helix, termed helix 8, in the C-terminal tail. Furthermore, these GPCRs were determined to possess several conserved residues in their transmembrane domains. The functional deficiencies of receptors in which these domains or residues have been mutated have not been examined in living cells due to their accumulation in the endoplasmic reticulum (ER), although the ligand affinities of these receptors have been tested in membrane preparations. Recent studies have demonstrated that ER-accumulated receptors are effectively exported from ER using membrane permeable ligands as pharmacological chaperones. Here, we identified several residues of the platelet-activating factor receptor and leukotriene B(4) type-II receptor that are crucial for export from ER. Moreover, we used their specific ligands as pharmacological chaperones to traffic ER-accumulated GPCRs to the cell surface in order to examine the functional deficiencies of each mutant receptor. Here, we introduce the novel technique of site-specific N-terminal labeling of cell surface proteins in living cells with Sortase-A, a transpeptidase isolated from Staphylococcus aureus, to evaluate the trafficking of receptors after agonist stimulation.
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Arachidonoyl-phosphatidylcholine oscillates during the cell cycle and counteracts proliferation by suppressing Akt membrane binding.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-28-2013
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The activity of protein kinase B (Akt)--a major kinase promoting cell proliferation and survival--oscillates during the cell cycle. To investigate whether membrane phospholipids may regulate Akt phosphorylation and thus activity, we monitored the lipid profile of nocodazole-synchronized mouse NIH 3T3 fibroblasts during the cell cycle by liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The proportion of sn-2-arachidonoyl-phosphatidylcholine (20:4-PC) inversely correlated with Akt activity. Increasing the cellular ratio of 20:4-PC by supplementation of 20:4-PC to the cell culture medium diminished Akt [serine (Ser)473] phosphorylation. Saturated and monounsaturated phosphatidylcholines, used as control had no effect; 20:4-PC reduced cell proliferation relative to controls, interfered with S-phase transition, and suppressed Akt downstream signaling and cyclin expression like LY294002, which is a specific inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. Additive effects of 20:4-PC and LY294002 were not observed, underlining the critical role of Akt for 20:4-PC signaling; 20:4-PC suppressed Akt membrane translocation as shown by immunofluorescence microscopy but left the concentration of the anchor lipid phosphatidylinositol-3,4,5-trisphosphate unchanged. An in vitro binding assay suggests that 20:4-PC attenuates the interaction of Akt with its membrane binding site. We conclude that 20:4-PC oscillates during the cell cycle and delays cell cycle progression by inhibiting Akt membrane binding.
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Human Fc?RIIA induces anaphylactic and allergic reactions.
Blood
PUBLISHED: 12-02-2011
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IgE and IgE receptors (Fc?RI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (Fc?RIIIA and Fc?RIV) expressed by neutrophils, rather than on IgE and Fc?RI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express Fc?RIIIA or Fc?RIV, but Fc?RIIA. We therefore investigated the possible role of Fc?RIIA in allergy by generating novel Fc?RIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, Fc?RIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking Fc?RIIA in vivo abolished these reactions. We identified mast cells to be responsible for Fc?RIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for Fc?RIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after Fc?RIIA engagement. IgG and Fc?RIIA may therefore contribute to allergic and anaphylactic reactions in humans.
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Polyunsaturated fatty acids are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransferase 3.
FASEB J.
PUBLISHED: 10-03-2011
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Long-chain polyunsaturated fatty acids (PUFAs) accumulate in mammalian testis during puberty and are essential for fertility. To investigate whether lysophospholipid acyltransferases determine the PUFA composition of testicular phospholipids during pubertal development, we compared their mRNA expression, in vitro activity, and specificity with the lipidomic profile of major phospholipids. The accumulation of PUFAs in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine correlated with an induced lysophosphatidic acid acyltransferase (LPAAT)3 mRNA expression, increased microsomal LPAAT3 activity, and shift of LPAAT specificity to PUFA-coenzyme A. LPAAT3 was induced during germ cell maturation, as shown by immunofluorescence microscopy. Accordingly, differentiation of mouse GC-2spd(ts) spermatocytes into spermatides up-regulated LPAAT3 mRNA, increased the amount of polyunsaturated phospholipids, and shifted the specificity for the incorporation of deuterium-labeled docosahexaenoic acid toward phosphatidylcholine and phosphatidylethanolamine. Stable knockdown of LPAAT3 in GC-2spd(ts) cells significantly decreased microsomal LPAAT3 activity, reduced levels of polyunsaturated phosphatidylethanolamine species, and impaired cell proliferation/survival during geneticin selection. We conclude that the induction of LPAAT3 during germ cell development critically contributes to the accumulation of PUFAs in testicular phospholipids, thereby possibly affecting sperm cell production.
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Analysis of two major intracellular phospholipases A(2) (PLA(2)) in mast cells reveals crucial contribution of cytosolic PLA(2)?, not Ca(2+)-independent PLA(2)?, to lipid mobilization in proximal mast cells and distal fibroblasts.
J. Biol. Chem.
PUBLISHED: 08-31-2011
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Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)?) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)?), and the role of cPLA(2)? in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)? (Pla2g4a(-/-)) or iPLA(2)? (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)?, not by iPLA(2)?, during Fc?RI-mediated activation and even during fibroblast-dependent maturation. Neither Fc?RI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)? from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)? in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)? is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.
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Phospholipase A2 superfamily members play divergent roles after spinal cord injury.
FASEB J.
PUBLISHED: 08-25-2011
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Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A(2) (PLA(2)) superfamily plays important roles in SCI. PLA(2) enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA(2) group IVA (cPLA(2) GIVA) and calcium-independent PLA(2) group VIA (iPLA(2) GVIA)], and a secreted form [secreted PLA(2) group IIA (sPLA(2) GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA(2)s play differing roles. cPLA(2) GIVA mediates protection, whereas sPLA(2) GIIA and, to a lesser extent, iPLA(2) GVIA are detrimental. Furthermore, completely blocking all three PLA(2)s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA(2) and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA(2) (sPLA(2) and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.
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International Union of Basic and Clinical Pharmacology. LXXXIV: leukotriene receptor nomenclature, distribution, and pathophysiological functions.
Pharmacol. Rev.
PUBLISHED: 07-21-2011
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The seven-transmembrane G protein-coupled receptors activated by leukotrienes are divided into two subclasses based on their ligand specificity for either leukotriene B(4) or the cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)). These receptors have been designated BLT and CysLT receptors, respectively, and a subdivision into BLT(1) and BLT(2) receptors and CysLT(1) and CysLT(2) receptors has been established. However, recent findings have also indicated the existence of putative additional leukotriene receptor subtypes. Furthermore, other ligands interact with the leukotriene receptors. Finally, leukotrienes may also activate other receptor classes, such as purinergic receptors. The aim of this review is to provide an update on the pharmacology, expression patterns, and pathophysiological roles of the leukotriene receptors as well as the therapeutic developments in this area of research.
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Prostaglandin E2-prostaglandin E receptor subtype 4 (EP4) signaling mediates UV irradiation-induced systemic immunosuppression.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-01-2011
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UV radiation induces systemic immunosuppression. Because nonsteroidal anti-inflammatory drugs suppress UV-induced immunosuppression, prostanoids have been suspected as a crucial mediator of this UV effect. However, the identity of the prostanoid involved and its mechanism of action remain unclear. Here, we addressed this issue by subjecting mice deficient in each prostanoid receptor individually or mice treated with a subtype-specific antagonist to UV irradiation. Mice treated with an antagonist for prostaglandin E receptor subtype 4 (EP4), but not those deficient in other prostanoid receptors, show impaired UV-induced immunosuppression, whereas administration of an EP4 agonist rescues the impairment of the UV-induced immunosuppression in indomethacin-treated mice. The EP4 antagonist treatment suppresses an increase in the number of CD4(+)/forkhead box P3-positive (Foxp3(+)) regulatory T cells (Treg cells) in the peripheral lymph nodes (LNs) and dendritic cells expressing DEC205 in the LNs and the skin after UV irradiation. Furthermore, the EP4 antagonist treatment down-regulates UV-induced expression of receptor activator of NF-?B ligand (RANKL) in skin keratinocytes. Finally, administration of anti-RANKL antibody abolishes the restoration of UV-induced immunosuppression by EP4 agonism in indomethacin-treated mice. Thus, prostaglandin E(2) (PGE(2))-EP4 signaling mediates UV-induced immunosuppression by elevating the number of Treg cells through regulation of RANKL expression in the epidermis.
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Cytosolic phospholipase A2alpha contributes to blood pressure increases and endothelial dysfunction under chronic NO inhibition.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 02-24-2011
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Nitric oxide (NO) is an important modulator of cardiovascular function. In this study, we examined whether cytosolic phospholipase A2? (cPLA2?), an initial enzyme in the arachidonic acid pathway, is involved in blood pressure (BP) elevation in a murine model of chronic NO inhibition.
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Role of leukotriene B4 in accelerated hyperlipidaemic renal injury.
Nephrology (Carlton)
PUBLISHED: 02-24-2011
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Glomerular infiltration of macrophages is a characteristic alteration of renal pathology in hyperlipidaemic renal injury. Leukotriene B4 (LTB4) is a bioactive eicosanoid and macrophage and has two key enzymes for LTB4 synthesis, 5-lipoxygenase and leukotriene A4 (LTA4) hydrolase. The purpose of this study was to evaluate the role of LTB4 in accelerated hyperlipidaemic renal injury.
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Mouse and human neutrophils induce anaphylaxis.
J. Clin. Invest.
PUBLISHED: 01-19-2011
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Anaphylaxis is a life-threatening hyperacute immediate hypersensitivity reaction. Classically, it depends on IgE, Fc?RI, mast cells, and histamine. However, anaphylaxis can also be induced by IgG antibodies, and an IgG1-induced passive type of systemic anaphylaxis has been reported to depend on basophils. In addition, it was found that neither mast cells nor basophils were required in mouse models of active systemic anaphylaxis. Therefore, we investigated what antibodies, receptors, and cells are involved in active systemic anaphylaxis in mice. We found that IgG antibodies, Fc?RIIIA and Fc?RIV, platelet-activating factor, neutrophils, and, to a lesser extent, basophils were involved. Neutrophil activation could be monitored in vivo during anaphylaxis. Neutrophil depletion inhibited active, and also passive, systemic anaphylaxis. Importantly, mouse and human neutrophils each restored anaphylaxis in anaphylaxis-resistant mice, demonstrating that neutrophils are sufficient to induce anaphylaxis in mice and suggesting that neutrophils can contribute to anaphylaxis in humans. Our results therefore reveal an unexpected role for IgG, IgG receptors, and neutrophils in anaphylaxis in mice. These molecules and cells could be potential new targets for the development of anaphylaxis therapeutics if the same mechanism is responsible for anaphylaxis in humans.
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The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-20-2010
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Tumors often are associated with a low extracellular pH, which induces a variety of cellular events. However, the mechanisms by which tumor cells recognize and react to the acidic environment have not been fully elucidated. T-cell death-associated gene 8 (TDAG8) is an extracellular pH-sensing G protein-coupled receptor that is overexpressed in various tumors and tumor cell lines. In this report, we show that TDAG8 on the surface of tumor cells facilitates tumor development by sensing the acidic environment. Overexpression of TDAG8 in mouse Lewis lung carcinoma (LLC) cells enhanced tumor development in animal models and rendered LLC cells resistant to acidic culture conditions by increasing activation of protein kinase A and extracellular signal-regulated kinase in vitro. Moreover, shRNA-mediated knockdown of endogenous TDAG8 in NCI-H460 human non-small cell lung cancer cells reduced cell survival in an acidic environment in vitro as well as tumor development in vivo. Microarray analyses of tumor-containing lung tissues of mice injected with TDAG8-expressing LLC cells revealed up-regulation of genes related to cell growth and glycolysis. These results support the hypothesis that TDAG8 enhances tumor development by promoting adaptation to the acidic environment to enhance cell survival/proliferation. TDAG8 may represent a therapeutic target for arresting tumor growth.
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Identification and characterization of a novel zebrafish semaphorin.
Neurosci. Lett.
PUBLISHED: 08-31-2010
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The semaphorin gene family contains numerous secreted and transmembrane proteins. Some of them function as the repulsive and attractive axon guidance molecules during development. Herein, we report the cloning and characterization of a novel member of zebrafish semaphorin gene, semaphorin 6E (sema6E). Sema6E is expressed predominantly in the nervous system during embryogenesis. Results also show that Sema6E binds Plexin-A1, but not other Plexins. Sema6E chemorepels not only dorsal root ganglion axons but also sympathetic axons. Therefore, Sema6E might utilize Plexin-A1 as a receptor to repel axons of the specific types during development.
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LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis.
Blood
PUBLISHED: 08-16-2010
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Lysophosphatidic acid (LPA) is a potent lipid mediator with a wide variety of biological actions mediated through G protein-coupled receptors (LPA(1-6)). LPA(4) has been identified as a G(13) protein-coupled receptor, but its physiological role is unknown. Here we show that a subset of LPA(4)-deficient embryos did not survive gestation and displayed hemorrhages and/or edema in many organs at multiple embryonic stages. The blood vessels of bleeding LPA(4)-deficient embryos were often dilated. The recruitment of mural cells, namely smooth muscle cells and pericytes, was impaired. Consistently, Matrigel plug assays showed decreased mural cell coverage of endothelial cells in the neovessels of LPA(4)-deficient adult mice. In situ hybridization detected Lpa4 mRNA in the endothelium of some vasculatures. Similarly, the lymphatic vessels of edematous embryos were dilated. These results suggest that LPA(4) regulates establishment of the structure and function of blood and lymphatic vessels during mouse embryogenesis. Considering the critical role of autotaxin (an enzyme involved in LPA production) and G?(13) in vascular development, we suggest that LPA(4) provides a link between these 2 molecules.
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Role of lysophosphatidic acid acyltransferase 3 for the supply of highly polyunsaturated fatty acids in TM4 Sertoli cells.
FASEB J.
PUBLISHED: 08-12-2010
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Sertoli cells supply germ cells with nutrients, including highly polyunsaturated fatty acids (hPUFAs), which are essential for testicular function. We have previously reported high expression of lysophosphatidic acid acyltransferase (LPAAT)3 in mature mouse testis and suggested an arachidonoyl-transferase activity to LPA. To investigate the role of LPAAT3 in the storage and release of PUFAs, TM4 Sertoli cells were stably transfected with LPAAT3-small hairpin (sh)RNA. Arachidonoyl-, eicosapentaenoyl-, and docosapentaenoyl-containing phosphatidylcholine (PC) and linoleoyl-containing phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylglycerol were significantly decreased as determined by liquid chromatography coupled to electrospray ionization mass spectrometry. Expression of murine LPAAT3 in Chinese hamster ovary (CHO)-K1 cells had essentially an opposite effect. The level of polyunsaturated PC correlated with cellular levels of free docosapentaenoic acid and eicosapentaenoic acid in TM4 and CHO-K1 cells, respectively. Activity assays using microsomal preparations as a source of LPAAT3 revealed an excessive PA synthesis from LPA acceptors for docosahexaenoyl-, arachidonoyl- and less pronounced for linoleoyl-CoA. We propose that the efficient incorporation of hPUFAs into PA-the precursor of several phospholipids, including PC-and the selective increase of the polyunsaturated PC pool in TM4 Sertoli cells might be required for the controlled release of hPUFAs and their supply to germ cells.
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Protective role of the leukotriene B4 receptor BLT2 in murine inflammatory colitis.
FASEB J.
PUBLISHED: 07-28-2010
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BLT2 is a low-affinity leukotriene B(4) (LTB(4)) receptor that is activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB(4). Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. To clarify the role of BLT receptors in intestinal inflammation, we assessed susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice lacking either BLT1 or BLT2. BLT2(-/-) mice exhibited increased sensitivity to DSS as compared to wild-type and BLT1(-/-) mice, with more severe body weight loss and inflammation. Expression of inflammatory cytokines such as interferon (IFN)-?, interleukin (IL)-1?, and IL-6, chemokines such as CXC chemokine ligand 9 (CXCL9) and C-C motif chemokine 19 (CCL19), and metalloproteinases was highly up-regulated in the colons of DSS-treated BLT2(-/-) mice, and there was an enhanced accumulation of activated macrophages. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also markedly accelerated in the crypts of DSS-treated BLT2(-/-) mice. Madin-Darby canine kidney II (MDCKII) cells transfected with BLT2 exhibited enhanced barrier function as measured by transepithelial electrical resistance (TER) and FITC-dextran leakage through MDCK monolayers. Thus, BLT2 is expressed in colon cryptic cells and appears to protect against DSS-induced colitis, possibly by enhancing barrier function in epithelial cells of the colon. These novel results suggest a direct anti-inflammatory role of BLT2 that is distinct from the proinflammatory roles of BLT1.
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Phosphorylation of lysophosphatidylcholine acyltransferase 2 at Ser34 enhances platelet-activating factor production in endotoxin-stimulated macrophages.
J. Biol. Chem.
PUBLISHED: 07-27-2010
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Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser(34) of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.
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Specific ligands as pharmacological chaperones: The transport of misfolded G-protein coupled receptors to the cell surface.
IUBMB Life
PUBLISHED: 05-27-2010
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In the endoplasmic reticulum (ER), quality control mechanisms distinguish between correctly and incorrectly folded structures to ensure that aberrant proteins are not processed along the secretory pathway. Numerous studies have demonstrated the functional rescue of ER-retained, aberrant proteins by small membrane permeable molecules called pharmacological chaperones. Pharmacological chaperones can bind to misfolded proteins, including G-protein coupled receptors (GPCRs), and promote their correct folding and export from the ER. Recently, common structural features of GPCRs have been uncovered, including the eighth helical domain in the C-terminal tail and conserved residues in the transmembrane domains. However, little is known about the importance of these features in signaling and intracellular trafficking, because receptors deficient in these domains are likely retained in the ER due to misfolding. In this review, we summarize the current knowledge about the requirement of these consensus domains and amino acid residues for the passing through the quality control of the ER. Furthermore, we propose the utilization of membrane permeable ligands for the transport of their cognate, ER-retained GPCRs to the cell surface. The chaperone activity of these ligands allows us to perform functional analyses of the structure-deficient receptors after their trafficking to the cell surface.
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Cytosolic phospholipase A2alpha mediates Pseudomonas aeruginosa LPS-induced airway constriction of CFTR -/- mice.
Respir. Res.
PUBLISHED: 04-29-2010
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Lungs of cystic fibrosis (CF) patients are chronically infected with Pseudomonas aeruginosa. Increased airway constriction has been reported in CF patients but underplaying mechanisms have not been elucidated.
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AML1 enhances the expression of leukotriene B4 type-1 receptor in leukocytes.
FASEB J.
PUBLISHED: 04-15-2010
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Leukotriene B(4) type-1 receptor (BLT1), which plays a role in various inflammatory diseases, is exclusively expressed in peripheral leukocytes, which suggests that its expression is stringently regulated. However, the precise mechanism of BLT1 expression is not fully understood. Here we report that acute myeloid leukemia 1 (AML1/Runx1) is involved in the enhancement of BLT1 expression in leukocytes. In retinoic acid (RA)-stimulated human promyelocytic leukemia (HL-60) cells, the transcription of the BLT1 gene was found to be significantly activated. RA did not directly modulate the BLT1 promoter, suggesting enhancers in other loci. DNase I-hypersensitivity analyses revealed an activated region, termed AE-BLex, at the intron-I:exon-II boundary. AE-BLex acts as an enhancer for the BLT1 promoter and possesses 2 AML1 recognition sites. The importance of AML1 was determined using electrophoretic mobility shift assays, reporter assays, and knockdown experiments. We demonstrated that the enhancement of BLT1 expression during the RA-induced differentiation of HL-60 cells is due to a loosening of the chromatin structure around AE-BLex, which leads to the incremental binding of AML1. The AML1/AE-BLex complex was confirmed in other BLT1- expressing leukemia cell lines and human peripheral leukocytes. Thus, AML1 enhances BLT1 expression by binding to AE-BLex, which is accessible in leukocytes.
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Role of PAF receptor in proinflammatory cytokine expression in the dorsal root ganglion and tactile allodynia in a rodent model of neuropathic pain.
PLoS ONE
PUBLISHED: 04-10-2010
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Neuropathic pain is a highly debilitating chronic pain following damage to peripheral sensory neurons and is often resistant to all treatments currently available, including opioids. We have previously shown that peripheral nerve injury induces activation of cytosolic phospholipase A(2) (cPLA(2)) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia, a hallmark of neuropathic pain. However, lipid mediators downstream of cPLA(2) activation to produce tactile allodynia remain to be determined.
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Intestinal anisakiasis can cause intussusception in adults: an extremely rare condition.
World J. Gastroenterol.
PUBLISHED: 04-10-2010
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We report an extremely rare case of adult intussusception caused by anisakiasis. A 41-year-old man was admitted into our hospital for right lower abdominal colicky pain. Ultrasonography and computed tomography revealed the presence of intussusception. As pneumo-dynamic resolution by colonoscopy failed, surgery was performed. The anisakis body was found in the submucosal layer of the resection specimen. The patient was discharged 9 d after the operation. Anisakiasis may cause intussusception in any country where sushi or sashimi now exists as a popular food. If suspicious, detailed clinical interview as to food intake prior to symptom development is crucial.
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Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1.
Nature
PUBLISHED: 03-11-2010
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Adiponectin is an anti-diabetic adipokine. Its receptors possess a seven-transmembrane topology with the amino terminus located intracellularly, which is the opposite of G-protein-coupled receptors. Here we provide evidence that adiponectin induces extracellular Ca(2+) influx by adiponectin receptor 1 (AdipoR1), which was necessary for subsequent activation of Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaMKKbeta), AMPK and SIRT1, increased expression and decreased acetylation of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), and increased mitochondria in myocytes. Moreover, muscle-specific disruption of AdipoR1 suppressed the adiponectin-mediated increase in intracellular Ca(2+) concentration, and decreased the activation of CaMKK, AMPK and SIRT1 by adiponectin. Suppression of AdipoR1 also resulted in decreased PGC-1alpha expression and deacetylation, decreased mitochondrial content and enzymes, decreased oxidative type I myofibres, and decreased oxidative stress-detoxifying enzymes in skeletal muscle, which were associated with insulin resistance and decreased exercise endurance. Decreased levels of adiponectin and AdipoR1 in obesity may have causal roles in mitochondrial dysfunction and insulin resistance seen in diabetes.
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[A case of intestinal tuberculosis associated with ileo-ileal fistula].
Nihon Shokakibyo Gakkai Zasshi
PUBLISHED: 03-06-2010
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A 25-year-old woman was admitted with fever and right lower abdominal pain that had started 3 days after the administration of antituberculosis agents for pulmonary tuberculosis. She was given a diagnosis of intestinal tuberculosis with ileo-ileal fistula formation on computed tomography, colonoscopy and laboratory test findings. She was kept on anti-tuberculosis agents. Soon after, ileus and a micro abscess appeared near the fistula. Ileocecal resection with partial ileal resection resulted in a good postoperative clinical course. Tuberculosis is still a major infectious disease. Intestinal tuberculosis is very rarely associated with fistula formation, and when it occurs, is difficult to differentiate from Crohn disease. Our case strongly suggests that typical intestinal tuberculosis findings can help in differential diagnosis between intestinal tuberculosis and Crohn disease.
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The leukotriene B4 receptor, BLT1, is required for the induction of experimental autoimmune encephalomyelitis.
Biochem. Biophys. Res. Commun.
PUBLISHED: 03-03-2010
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Leukotriene B(4) (LTB(4)) is a potent chemoattractant and activator of neutrophils, macrophages and T cells. These cells are a key component of inflammation and all express BLT1, a high affinity G-protein-coupled receptor for LTB(4). However, little is known about the neuroimmune functions of BLT1. In this study, we describe a distinct role for BLT1 in the pathology of experimental autoimmune encephalomyelitis (EAE) and T(H)1/T(H)17 immune responses. BLT1 mRNA was highly upregulated in the spinal cord of EAE mice, especially during the induction phase. BLT1(-/-) mice had delayed onset and less severe symptoms of EAE than BLT1(+/+) mice. Additionally, inflammatory cells were recruited to the spinal cord of asymptomatic BLT1(+/+), but not BLT1(-/-) mice before the onset of disease. Ex vivo studies showed that both the proliferation and the production of IFN-gamma, TNF-alpha, IL-17 and IL-6 were impaired in BLT1(-/-) cells, as compared with BLT1(+/+) cells. Thus, we suggest that BLT1 exacerbates EAE by regulating the migration of inflammatory cells and T(H)1/T(H)17 immune responses. Our findings provide a novel therapeutic option for the treatment of multiple sclerosis and other T(H)17-mediated diseases.
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Decrease in expression or activity of cytosolic phospholipase A2alpha increases cyclooxygenase-1 action: A cross-talk between key enzymes in arachidonic acid pathway in prostate cancer cells.
Biochim. Biophys. Acta
PUBLISHED: 02-26-2010
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The eicosanoid pathway is activated in many types of cancers including prostate. Eicosanoids are synthesized from intracellular arachidonic acid (AA), which is released from membrane glycerophospholipids mainly by the action of cytosolic phospholipase A(2)alpha (cPLA(2)alpha). Thus, targeting cPLA(2)alpha has been proposed as a treatment option. The aim of this study was to determine the effect of cPLA(2)alpha inhibition on cyclooxygenase (COX) expression and PGE(2) production. Inhibition of cPLA(2)alpha expression by siRNA or activity by Efipladib in prostate cancer cell lines (PC3 and LNCaP) led to an increase in COX-1 protein and PGE(2) levels in a dose-dependent manner from 24 to 72 h. The COX-2 response was less evident. Efipladib treatment increased COX-1 promoter transcriptional activity without changing the rate of COX-1 protein degradation. Treatment with Efipladib also led to a decrease in most LOX products (HETEs) as measured by LC/MS/MS. Replenishing 5- and 12-HETEs abolished Efipladib-induced COX-1 and PGE(2) levels. Decreasing 5- and 12-HETE production, as a result of treating cells with inhibitors MK886 and Baicalein, respectively, mimicked the effect of Efipladib on COX-1 and PGE(2) levels. Hence, the mechanism underlying the cPLA(2)alpha inhibition-induced COX-1 is likely due to a decrease in LOX products, which may exert a negative feedback on COX-1 gene expression in prostate cancer cells. Considering that PGE(2) is a potent promoter of cancer cell proliferation and survival, understanding the mechanism coupling cPLA(2)alpha with COX-1 is of potential clinical significance.
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Expression, purification and characterization of leukotriene B(4) receptor, BLT1 in Pichia pastoris.
Protein Expr. Purif.
PUBLISHED: 02-13-2010
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The high yield expression of BLT1, a G-protein coupled receptor for leukotriene B(4), was established in Pichia pastoris for structural studies. Guinea pig BLT1 was expressed in a functional form without post-translational modifications for the rapid purification and the crystallization. Among the BLT1s from four species, only guinea pig BLT1 was successfully expressed with the comparable binding affinity to BLT1 of native guinea pig tissues for several ligands. Only Asn4 of the two putative N-glycosylation sites was glycosylated, and the mutation to Ala to avoid glycosylation did not affect the ligand binding affinity. However, the N-terminal region of the mutant was digested at the carboxyl ends of Arg3 and Arg8, as detected by N-terminal amino acid sequencing, and Ser309 in the C-terminal region was partially phosphorylated, as identified in the micro-sequencing by Q-TOF-MS/MS. To avoid chemical heterogeneity, the N-terminal peptide (1-14) truncated and the C-terminal phosphorylation-site eliminated mutant was generated. The binding affinity of the mutants membrane fraction for LTB(4) was K(d)=6.6 nM and B(max)=50.0 pmol/mg membrane protein. The yield of purified mutant was approximately 0.3-0.4 mg from 1L culture, and the protein showed a single peak at molecular weight of 100 kDa in gel-filtration and no glycosylation or phosphorylation in MALDI-TOF MS.
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Lipid signaling in cytosolic phospholipase A2alpha-cyclooxygenase-2 cascade mediates cerebellar long-term depression and motor learning.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-28-2010
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In this study, we show the crucial roles of lipid signaling in long-term depression (LTD), that is, synaptic plasticity prevailing in cerebellar Purkinje cells. In mouse brain slices, we found that cPLA(2)alpha knockout blocked LTD induction, which was rescued by replenishing arachidonic acid (AA) or prostaglandin (PG) D(2) or E(2). Moreover, cyclooxygenase (COX)-2 inhibitors block LTD, which is rescued by supplementing PGD(2)/E(2). The blockade or rescue occurs when these reagents are applied within a time window of 5-15 min following the onset of LTD-inducing stimulation. Furthermore, PGD(2)/E(2) facilitates the chemical induction of LTD by a PKC activator but is unable to rescue the LTD blocked by a PKC inhibitor. We conclude that PGD(2)/E(2) mediates LTD jointly with PKC, and suggest possible pathways for their interaction. Finally, we demonstrate in awake mice that cPLA(2)alpha deficiency or COX-2 inhibition attenuates short-term adaptation of optokinetic eye movements, supporting the view that LTD underlies motor learning.
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The endocannabinoid 2-arachidonoylglycerol produced by diacylglycerol lipase alpha mediates retrograde suppression of synaptic transmission.
Neuron
PUBLISHED: 01-16-2010
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Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGLalpha) and beta (DGLbeta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLalpha knockout mice, whereas the retrograde suppression was intact in DGLbeta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLalpha knockout brains, whereas the 2-AG content was normal in DGLbeta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLalpha, but not by DGLbeta, mediates retrograde suppression at central synapses.
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Liquid chromatography/mass spectrometry analysis revealing preferential occurrence of non-arachidonate-containing phosphatidylinositol bisphosphate species in nuclei and changes in their levels during cell cycle.
Rapid Commun. Mass Spectrom.
PUBLISHED: 01-14-2010
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Phosphatidylinositol phosphates (PtdInsPs) are present within the nucleus, as well as in the membrane. In this mass spectrometry study, different acyl-containing species of endonuclear PtdInsPs were analyzed in order to clearly understand the role of individual molecular species. A (34:1) acyl-containing phosphatidylinositol bisphosphate [PtdInsP(2)(34:1)] and PtdInsP(2)(36:1) were preferentially detected in envelope-less nuclei prepared from various cultured human cells, while PtdInsP(2)(38:4) was not a major component within these nuclei. A significant amount of PtdInsP(2)(34:0) was detected in the HeLa cell nucleus, but not in the A431 and THP-1 cell nuclei. During the cell cycle in HeLa cells, PtdInsP(2)(34:0) levels increased in the early G1 phase, and then gradually decreased through S phase, while PtdInsP(2)(34:1) levels tended to decrease only in late G1 phase and PtdInsP(2)(38:4) did not change significantly. Thus, individual PtdInsP(2) species apparently play different roles in nuclear events based on individual regulation of endonuclear levels. The non-arachidonate-containing species were also detected in normal human blood and fluids, suggesting that these minor species may have unique functions in the human body. The techniques used in this study will be applied to clinical studies on a PtdInsPs metabolism.
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Amino acid residues critical for endoplasmic reticulum export and trafficking of platelet-activating factor receptor.
J. Biol. Chem.
PUBLISHED: 12-10-2009
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Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.
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Targeted lipidomics reveals mPGES-1-PGE2 as a therapeutic target for multiple sclerosis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-07-2009
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The arachidonic acid (AA) cascade produces eicosanoids, such as prostaglandins (PGs), that regulate physiological and pathological functions. Although various nonsteroidal anti-inflammatory drugs have been developed, blocking upstream components (cyclooxygenase-1 and -2) of the AA cascade leads to severe side effects, including gastrointestinal ulcers and cardiovascular events, respectively, due to the complexity of the AA cascade. Here, using an AA cascade-targeted lipidomics approach, we report that microsomal PGE synthase 1 (mPGES-1) plays a key role in experimental autoimmune encephalomyelitis (EAE). Eicosanoids (mainly PGD(2)) are produced constitutively in the spinal cord of naive mice. However, in EAE lesions, the PGE(2) pathway is favored and the PGD(2), PGI(2), and 5-lipoxygenase pathways are attenuated. Furthermore, mPGES-1(-/-) mice showed less severe symptoms of EAE and lower production of IL-17 and IFN-gamma than mPGES-1(+/+) mice. Expression of PGE(2) receptors (EP1, EP2, and EP4) was elevated in EAE lesions and correlated with clinical symptoms. Immunohistochemistry on central nervous systems of EAE mice and multiple sclerosis (MS) patients revealed overt expression of mPGES-1 protein in microglia/macrophages. Thus, the mPGES-1-PGE(2)-EPs axis of the AA cascade may exacerbate EAE pathology. Our findings have important implications for the design of therapies for MS.
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A distinctive role of the leukotriene B4 receptor BLT1 in osteoclastic activity during bone loss.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-24-2009
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Although leukotriene B(4) (LTB(4)) is produced in various inflammatory diseases, its functions in bone metabolism remain unknown. Using mice deficient in the high-affinity LTB(4) receptor BLT1, we evaluated the roles of BLT1 in the development of two bone resorption models, namely bone loss induced by ovariectomy and lipopolysaccharide. Through observations of bone mineral contents and bone morphometric parameters, we found that bone resorption in both models was significantly attenuated in BLT1-deficient mice. Furthermore, osteoclasts from BLT1-deficient mice showed reduced calcium resorption activities compared with wild-type osteoclasts. Osteoclasts expressed BLT1, but not the low-affinity LTB(4) receptor BLT2, and produced LTB(4). LTB(4) changed the cell morphology of osteoclasts through the BLT1-Gi protein-Rac1 signaling pathway. Given the causal relationship between osteoclast morphology and osteoclastic activity, these findings suggest that autocrine/paracrine LTB(4) increases the osteoclastic activity through the BLT1-Gi protein-Rac1 signaling pathway. Inhibition of BLT1 functions may represent a strategy for preventing bone resorption diseases.
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Transcriptional regulation of human G2A in monocytes/ macrophages: involvement of c/EBPs, Runx and Pu.1.
Genes Cells
PUBLISHED: 11-20-2009
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G2 accumulation (G2A) is a G-protein coupled receptor, activated by several ligands and stimuli, such as lysophosphatidylcholine (LPC), extracellular low pH and oxidized phospholipids including 9- and 13-hydroxyoctadecadienoic acid, and has been implicated in mediating inflammatory process under oxidative conditions. Recently, it was demonstrated that G2A in monocytes/macrophages plays critical roles in atherosclerosis deterioration, and therefore its transcriptional regulation in monocytes/macrophages is of great interest. Here, we first confirmed the expression of human G2A (hG2A) in lymph nodes, spleen and peripheral blood leukocytes, including monocytes. Thereafter, transcription start site (TSS) of hG2A was determined by 5-rapid amplification of cDNA ends analysis. In the course of the analysis, we found that two transcriptional variants, hG2A-a and -b, are produced by alternative splicing, resulting in the production of N-terminal different hG2A proteins with similar sensitivity to low pH and LPC. Using a monocytic cell line THP-1 as a model, transcription of hG2A was precisely examined, and we demonstrated that it is dependent both on the chromatin structure around TSS, and on the binding of the transcription factors (c/EBPalpha and beta, Runx1 and Pu.1) to their cis-elements, located at the core promoter just upstream of TSS.
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Separation and quantification of sn-1 and sn-2 fatty acid positional isomers in phosphatidylcholine by RPLC-ESIMS/MS.
J. Biochem.
PUBLISHED: 10-30-2009
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Endogenous phosphatidylcholine in biological membranes exists as isomers with acyl moieties at the sn-1 or sn-2 positions of the glycerol backbone. However, detailed biochemical information on these positional isomers is not generally available. This study is the first report on the separation and identification of positional isomers of endogenous phosphatidylcholine using reversed-phase LC-ESIMS/MS. The separation of positional isomers in PC was achieved by using ultra performance LC, which uses a high-resolution HPLC system. To identify positional isomers in individual PC species, their lyso-PC-related fragments and fatty acids, which were obtained by MS/MS analysis in the negative ion mode, were used. From the application results of biological samples, the lipid extracts of mouse brain were found to be abundant in PC containing 22:6 at the sn-1 position of the glycerol backbone. However, the lipid extracts from mouse heart and liver were not abundant in positional isomers. This achievement demonstrates that the relative amounts of positional isomers in various tissues or molecular species differ. These results will be useful for the clarification of the biological mechanisms of remodelling enzymes such as phospholipase and acyltransferase. Thus, our report provides a novel and critical milestone in understanding how molecular composition of phospholipids is established and their biological roles.
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Attenuated Th1 induction by dendritic cells from mice deficient in the leukotriene B4 receptor 1.
Biochimie
PUBLISHED: 09-25-2009
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Dendritic cells (DCs) are important antigen-presenting cells that control Th1- and Th2-type immunological reactions by releasing cytokines and interacting directly with T cells. Leukotriene B4 (LTB4), a classical proinflammatory lipid mediator for phagocytes, was recently identified as an important attractant for effector CD4(+) and CD8(+) T cells. However, little information is available on the roles of LTB4 and its receptor BLT1 in DCs. Here we show that functional BLT1 expressed in mouse bone marrow-derived DCs (BMDCs) plays important role in initiating Th1-type immune response. Detailed analyses using BMDCs revealed that BLT1-deficient DCs produced less IL-12p70 than WT DCs, leading to attenuated IFN-gamma production in an allogeneic mixed lymphocyte reaction. Adoptive transfer of antigen-loaded BLT1-deficient DCs into naïve WT mice induced a weakened Th1- and enhanced Th2-response in vivo compared to WT DCs. BLT1-deficient mice consistently showed much attenuated delayed-type hypersensitivity (DTH), in which Th1-type cellular responses play a key role, and popliteal lymph node cells of BLT1-deficient mice showed reduced production of Th1 cytokines after DTH induction compared to cells from WT mice. Thus, in addition to its role in inflammation, the LTB4-BLT1 axis is important in initiating Th1-type immunological reactions mediated by DCs.
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Prostaglandin F(2alpha) receptor signaling facilitates bleomycin-induced pulmonary fibrosis independently of transforming growth factor-beta.
Nat. Med.
PUBLISHED: 09-14-2009
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Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by fibroblast proliferation and excess deposition of collagen and other extracellular matrix (ECM) proteins, which lead to distorted lung architecture and function. Given that anti-inflammatory or immunosuppressive therapy currently used for IPF does not improve disease progression therapies targeted to blocking the mechanisms of fibrogenesis are needed. Although transforming growth factor-beta (TGF-beta) functions are crucial in fibrosis, antagonizing this pathway in bleomycin-induced pulmonary fibrosis, an animal model of IPF, does not prevent fibrosis completely, indicating an additional pathway also has a key role in fibrogenesis. Given that the loss of cytosolic phospholipase A(2) (cPLA(2)) suppresses bleomycin-induced pulmonary fibrosis, we examined the roles of prostaglandins using mice lacking each prostoaglandin receptor. Here we show that loss of prostaglandin F (PGF) receptor (FP) selectively attenuates pulmonary fibrosis while maintaining similar levels of alveolar inflammation and TGF-beta stimulation as compared to wild-type (WT) mice, and that FP deficiency and inhibition of TGF-beta signaling additively decrease fibrosis. Furthermore, PGF(2alpha) is abundant in bronchoalveolar lavage fluid (BALF) of subjects with IPF and stimulates proliferation and collagen production of lung fibroblasts via FP, independently of TGF-beta. These findings show that PGF(2alpha)-FP signaling facilitates pulmonary fibrosis independently of TGF-beta and suggests this signaling pathway as a therapeutic target for IPF.
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Demethylallosamidin, a chitinase inhibitor, suppresses airway inflammation and hyperresponsiveness.
Biochem. Biophys. Res. Commun.
PUBLISHED: 09-06-2009
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Acidic mammalian chitinase is upregulated in response to allergen exposure in the lung. We investigated the effects of chitinase inhibitors, allosamidin (Allo) and demethylallosamidin (Dma), on asthmatic responses. Mice were subjected to IL-13 instillation into the airways or to ovalbumin sensitization plus exposure with or without treatment of Allo or Dma. Airway hyperresponsiveness (AHR) and inflammation were evaluated. Allo and Dma attenuated airway eosinophilia and the upregulation of eotaxin after IL-13 instillation, while Dma, but not Allo, suppressed AHR in IL-13-induced asthma. Allo or Dma suppressed the elevated chitinase activity in BAL fluids after IL-13 to similar levels. The bronchoprotective PGE(2) levels in BAL fluids were elevated after IL-13 instillation. Allo, but not Dma, suppressed the overproduction of PGE(2) and the expression of COX-2 and PGE synthase-1 induced by IL-13. In ovalbumin-induced asthma, Dma suppressed AHR more strongly than Allo. These findings suggest that Dma attenuates asthmatic responses induced by IL-13 without affecting PGE(2) synthesis. Dma may have potential as therapeutic agents for asthma.
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Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis.
J. Lipid Res.
PUBLISHED: 07-08-2009
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Glycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain putative acyltransferase motifs, but their biochemical properties and physiological roles are not completely understood. Here, we demonstrated that mouse LPAAT3, previously known as mouse AGPAT3, possesses strong LPAAT activity and modest lysophosphatidylinositol acyltransferase activity with a clear preference for arachidonoyl-CoA as a donor. This enzyme is highly expressed in the testis, where CDP-diacylglycerol synthase 1 preferring 1-stearoyl-2-arachidonoyl-phosphatidic acid as a substrate is also highly expressed. Since 1-stearoyl-2-arachidonoyl species are the main components of phosphatidylinositol, mouse LPAAT3 may function in both the de novo and remodeling pathways and contribute to effective biogenesis of 1-stearoyl-2-arachidonoyl-phosphatidylinositol in the testis. Additionally, the expression of this enzyme in the testis increases significantly in an age-dependent manner, and beta-estradiol may be an important regulator of this enzymes induction. Our findings identify this acyltransferase as an alternative important enzyme to produce phosphatidylinositol in the testis.
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Update of the LIPID MAPS comprehensive classification system for lipids.
J. Lipid Res.
PUBLISHED: 06-06-2009
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In 2005, the International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium developed and established a "Comprehensive Classification System for Lipids" based on well-defined chemical and biochemical principles and using an ontology that is extensible, flexible, and scalable. This classification system, which is compatible with contemporary databasing and informatics needs, has now been accepted internationally and widely adopted. In response to considerable attention and requests from lipid researchers from around the globe and in a variety of fields, the comprehensive classification system has undergone significant revisions over the last few years to more fully represent lipid structures from a wider variety of sources and to provide additional levels of detail as necessary. The details of this classification system are reviewed and updated and are presented here, along with revisions to its suggested nomenclature and structure-drawing recommendations for lipids.
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Recent progress on acyl CoA: lysophospholipid acyltransferase research.
J. Lipid Res.
PUBLISHED: 06-06-2009
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Cells of all organisms are enclosed by a plasma membrane containing bipolar lipids, cholesterol, and proteins. Cellular membranes contain several classes of glycerophospholipids, which have numerous structural and functional roles in cells. Polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid are usually located at the sn-2 position, but not the sn-1 position, of glycerophospholipids in an asymmetrical manner. Glycerophospholipids are first formed by the de novo pathway (Kennedy pathway) using acyl-CoAs as donors. Subsequently, in the remodeling pathway (Lands cycle), cycles of deacylation and reacylation of glycerophospholipids modify the fatty acid composition to generate mature membrane with asymmetry and diversity. Both pathways were proposed in the 1950s. Whereas the enzymes involved in the Kennedy pathway have been well characterized, little is known about the enzymes involved in the Lands cycle. Recently, several laboratories, including ours, have identified enzymes working in the Lands cycle from the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family, and also from the membrane bound O-acyltransferases (MBOAT) family. These discoveries have prompted a robust surge of research in this field. In this review, we focus on the cloning and characterization of lysophospholipid acyltransferases (LPLATs), which contribute to membrane asymmetry and diversity.
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Analysis of oxidized phosphatidylcholines as markers for oxidative stress, using multiple reaction monitoring with theoretically expanded data sets with reversed-phase liquid chromatography/tandem mass spectrometry.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 05-12-2009
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Recently, it was reported that oxidized phosphatidylcholine shows biological activities via scavenger receptor CD36 or Toll-like receptor 4 (TLR4)-TRIF. Thus, the analysis of oxidized phospholipids is essential in understanding these biological roles. Here, we report an analytical method for oxidized phosphatidylcholines using multiple reaction monitoring (MRM) with theoretically expanded data sets. This analytical method was performed by a quadrupole linear ion trap mass spectrometer with ultra performance LC (UPLC). To investigate whether this established analytical method was applicable to biological samples, we performed variation analysis of oxidized PCs using a myocardial ischemia-reperfusion model. Most oxidized PCs were detected in higher amounts in the ischemic myocardium than in the non-ischemic myocardium. From these application results, this established method is a valuable tool for the global analysis of oxidized PCs. In the future, our study can provide further understanding of how oxidized phospholipids are produced and are correlated to various diseases.
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Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation.
Annu. Rev. Pharmacol. Toxicol.
PUBLISHED: 04-28-2009
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Prostaglandins, leukotrienes, platelet-activating factor, lysophosphatidic acid, sphingosine 1-phosphate, and endocannabinoids, collectively referred to as lipid mediators, play pivotal roles in immune regulation and self-defense, and in the maintenance of homeostasis in living systems. They are produced by multistep enzymatic pathways, which are initiated by the de-esterification of membrane phospholipids by phospholipase A2s or sphingo-myelinase. Lipid mediators exert their biological effects by binding to cognate receptors, which are members of the G protein-coupled receptor superfamily. The synthesis of the lipid mediators and subsequent induction of receptor activity is tightly regulated under normal physiological conditions, and enzyme and/or receptor dysfunction can lead to a variety of disease conditions. Thus, the manipulation of lipid mediator signaling, through either enzyme inhibitors or receptor antagonists and agonists, has great potential as a therapeutic approach to disease. In this review, I summarize our current state of knowledge of the synthesis of lipid mediators and the function of their cognate receptors, and discuss the effects of genetic or pharmacological ablation of enzyme or receptor function on various pathophysiological processes.
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Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.
J. Biol. Chem.
PUBLISHED: 04-22-2009
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p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA4. Here we report that p2y5 is a novel LPA receptor coupling to the G13-Rho signaling pathway. "LPA receptor-null" RH7777 and B103 cells exogenously expressing p2y5 showed [3H]LPA binding, LPA-induced [35S]guanosine 5-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and Gs/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA6.
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Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1.
J. Lipid Res.
PUBLISHED: 04-21-2009
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Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys(211)) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids.
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The cyclooxygenase-2 selective inhibitor, etodolac, but not aspirin reduces neovascularization in a murine ischemic hind limb model.
Eur. J. Pharmacol.
PUBLISHED: 04-06-2009
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Cyclooxygenase inhibitors are often prescribed to relieve severe ischemic leg pain in critical ischemic limb patients. Prescription of high doses of aspirin and selective cyclooxygenase-2 inhibitors is reported to increase cardiovascular events through suppression of the vasodilative prostanoid prostaglandin I(2) in endothelium. Here, we evaluated the influence of aspirin and etodolac, a selective cyclooxygenase-2 inhibitor, on neovascularization using a murine ischemia hind limb model. C57BL/6J mice were treated with aspirin or etodolac for twenty-eight days after induction of ischemia. We exploited a concentration of the agents that suppressed cyclooxygenase activity efficiently, especially in prostaglandin I(2) production. Recovery of limb blood perfusion and capillary density in ischemic limbs was significantly suppressed by etodolac treatment when compared to the aspirin treated group and untreated group. Production of 6-keto prostaglandin F(1alpha) and prostaglandin E(2) was lower in the aspirin treated group when compared with the etodolac-treated group. Also, these concentrations were lower in both treatment groups compared with the untreated group. Immunohistochemical analysis suggested cyclooxygenase-2 was expressed in endothelium but not in inflammatory cells in ischemic tissue from the acute to chronic phase. Cyclooxygenase-1 was expressed strongly in inflammatory cells in the acute phase. Furthermore, bone marrow-derived mononuclear cell transplantation improved neovascularization, whereas aspirin and etodolac did not inhibit these effects. Production of arachidonic acid metabolites by transplanted cells was independent of the improvement of neovascularization. In conclusion, cyclooxygenase-2 inhibition reduces ischemia-induced neovascularization.
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Global analysis of triacylglycerols including oxidized molecular species by reverse-phase high resolution LC/ESI-QTOF MS/MS.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 03-26-2009
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Recently, global analysis of triacylglycerols (TAGs) has become increasingly important in studies of abnormality of lipid metabolism in metabolic syndrome. TAGs consist of various molecular species, caused by their three fatty acyl chains with a large variety of carbon chain lengths and degrees of unsaturation. Therefore, most previously reported methods have been insufficient in global detection of TAGs including their structural isomers and TAGs with oxidized or odd number acyl carbon chain. Here we report an effective method for global analysis of TAG molecular species from complex lipid mixtures of mouse liver and white adipose tissue (WAT) using reverse-phased high resolution liquid chromatography (LC) coupled with electrospray ionization (ESI)-quadrapole/time of flight hybrid mass spectrometer (QTOF-MS). For effective profiling of TAG molecular species, sensitive two-dimensional (2D) maps were constructed and individual structures were correctly identified by the elution profile and MS/MS. As a result, TAGs including their structural isomers and TAGs with an odd number acyl carbon chain were separated and detected effectively on the 2D map as compared with conventional high performance LC. It was also found that our 2D profiling method was useful in searching characteristic molecular species globally. In mouse WAT, novel oxidized TAGs, which were mainly formed by hydroperoxidation of one of their linoleic acyl chains, were effectively detected in comparison with TAG molecular species of mouse liver.
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