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Functional analysis of Pseudomonas ceramidase
Abstracts / Chemistry and Physics of Lipids 149S (2007) S56–S73
mutations render D-bifunctional protein deficiency as a protein folding disease. Latest results on these experiments will be discussed. Reference Ferdinandusse, Ylianttila, Gloerich, Koski, Oostheim, Waterham, Hiltunen, Wanders, Glumoff, 2006. Am. J. Hum. Genet. 78, 112–124.
doi:10.1016/j.chemphyslip.2007.06.149 PO 100
S67
imply that PAF is a putative intracellular mediator of IL-1 actions on mesangial cells and the inhibition of IL-1-elicited synthesis of PAF by OOPLE may confer protection to renal inflammation. Acknowledgement: The project is co-funded by the European Social Fund and National Resources. doi:10.1016/j.chemphyslip.2007.06.150 PO 101
Functional analysis of Pseudomonas ceramidase Nozomu Okino 1 , Makoto Ito 2 1 Department
IL-1 induces a rapid and transient increase of intracellular platelet activating factor in a human mesangial cell line—Modulation by an olive oil polar lipid extract
of Bioscience and Biotechnology, Kyushu University, Japan; 2 Department of Bioscience and Biotechnology, Kyushu University Bio-Architecture Center, Kyushu University, Japan
Tzortzis Nomikos, Elizabeth Fragopoulou, Eleftheria Rekliti, Haralabos Karantonis, Smaragdi Antonopoulou
Ceramidase (CDase) is an enzyme that catalyzes the hydrolysis of the N-acyl linkage of ceramide to generate fatty acid and sphingosine base. We previously reported the purification, molecular cloning and characterization of a neutral CDase from Pseudomonas aeruginosa strain AN17. Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (PlcH) in the genome of P. aeruginosa, which is a well-known pathogen for opportunistic infections. Recently, we found that the simultaneous production of PlcH and CDase was induced by several lipids, and PlcH-induced hemolysis was significantly enhanced by the action of CDase. When the strain was cultured with sphingomyelin or phosphatidylcholine, which is abundant in plasma membranes of mammalian cells, the production of both enzymes drastically increased, causing an increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of CDase but not PlcH. To further investigate the functional role of CDase, the gene expression of CDase was examined by RT-PCR. When the strain was cultured with sphingomyelin, which is the most effective lipid for the induction of CDase, the mRNA level of CDase was significantly elevated. These results may indicate that CDase, which is induced by plasma membrane lipids, enhances PlcH-induced cytotoxicity and provides new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.
Department of Science of Nutrition-Dietetics, Harokopio University, Greece Platelet activating factor (PAF) is a potent phospholipid inflammatory mediator implicated in the pathogenesis of glomerulosclerosis. IL-1 demonstrates a plethora of actions on human mesangial cells regulating the inflammatory response and matrix turnover within the mesangium. Olive oil possesses potent antiinflammatory properties and previous studies have shown that an olive oil polar lipid extract (OOPLE) is able to attenuate the formation of early atherosclerotic lesions in diet-induced hypercholesterolemic rabbits. Glomerulosclerosis and atherosclerosis share common histological and biochemical characteristics, therefore the objective of this study was to investigate the effect of IL-1 on PAF production by a human mesangial cell line (HMCL) and the modulation of this action by the OOPLE. IL-1 induced a dose- and time-dependent increase of intracellular PAF synthesis while no PAF was detected in the extracellular medium. The IL-1 (1ng/ml)-induced intracellular PAF elevation (2–3-fold) peaked 30 min after the stimulation, partly via activation of the lyso-PAF acetyltransferase of the remodeling biosynthetic pathway and returned to basal levels in an hour. Both basal and IL-1-stimulated PAF levels were increased by the presence of pefabloc due to PAFAH inhibition. Preincubation of HMCL with OOPLE (20–2200 ng/ml) resulted in a dose dependent inhibition of IL-1-induced PAF increase while co-incubation of IL-1 with OOPLE slightly attenuated the activation of PAF synthesis. In conclusion, the above results
S68
Abstracts / Chemistry and Physics of Lipids 149S (2007) S56–S73
References Okino, N., et al., 1998. J. Biol. Chem. 273, 14368–14373. Okino, N., et al., 1999. J. Biol. Chem. 274, 36616–36622. Okino, N., Ito, M., 2007. J. Biol. Chem. 282, 6021–6030.
it may play an important role in the control of human serum lipid/lipoprotein balance. doi:10.1016/j.chemphyslip.2007.06.152 PO 103
doi:10.1016/j.chemphyslip.2007.06.151 PO 102
OSBP-related protein 10 (ORP10), a novel regulator of lipid homeostasis Julia Perttil¨a, Krista Merikanto, Jussi Naukkarinen, Nicolas Martin, Vesa M. Olkkonen, Leena Peltonen Department of Molecular Medicine, National Public Health Institute, Finland The oxysterol-binding-protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans and are implicated in a number of vital biological functions. The role of different ORP genes and corresponding proteins in human lipid metabolism has so far not been addressed. We used genetics to study the potential role of ORP genes in regulation of human serum lipids. We tested linkage of serum lipid levels to three ORP genes (ORP2, ORP9 and ORP10) by genotyping 25 HapMap SNPs tagging their allelic diversity in 92 Finnish dyslipidemia families (ascertained for low-HDL or FCHL) with a total of 1070 individuals with well characterized serum lipid profiles. Only one of the tested ORP genes, ORP10, provided evidence for linkage both when using 5% extreme lipid levels as affected in the dichotomized linkage analysis and in the quantitative analysis using MERLIN software. Multiple SNPs of the ORP10 gene revealed evidence for linkage to the HDL and triglyceride levels (high triglycerides LODmax = 2.405 with SNP rs9853939, quantitative linkage to HDL levels LOD = 2.01, P = 0.0012 with SNP rs6807471). These results imply that allelic variants of the gene encoding ORP10 may contribute to triglyceride and HDL levels in Finnish dyslipidemic families. To obtain functional evidence for this hypothesis, we studied the effects of ORP10 silencing by short interfering siRNA on cellular lipid biosyntheses in the Huh7 (human hepatoma) cells. Knock-down of ORP10 caused a significant increase in the synthesis of cholesterol and triglycerides. Analysis by real-time RTPCR quantification revealed that ORP10 knock-down leads to an increase in the mRNA for SREBP-2, a major transcriptional regulator of cellular lipid homeostasis, suggesting that ORP10 impacts on lipids at the level of gene expression. These results identify ORP10 as a novel modulator of cellular lipid homeostasis and suggest that
Participation of the glycosphingolipid biosynthesis in the differentiation of renal collecting duct cells Lucila Pescio, Francisco Leocata-Nieto, Nicol´as Favale, Mar´ıa del Carmen Fernandez-Tom´e, Norma Sterin-Speziale Department of Biological Sciences, University of Buenos Aires, Argentina MDCK is a non-transformed renal collecting cellular line that conserves the capacity to differentiate in particular conditions of cell culture. To differentiate, collecting duct cells have to acquire a polarized phenotype. Cell polarization is characterized by the presence of apical and basolateral membrane domains which differ in their protein content as well as in lipid composition. While glycosphingolipids (GSL) predominate in the apical membrane, sphingomyelin is the major sphingolipid of the basolateral membrane. It has been previously reported that extracellular hypertonicity induces cell polarization/differentiation, but the mechanism involved is not well understood. In this study, we have examined the role of the GSL biosynthesis in the process of collecting duct cell differentiation. Confluent MDCK cells were submitted to high NaCl concentration (250 mM) for 24 and 48 h. Glycosphingolipid metabolism was determined by using 14 C-galactose as radioactive precursor. The cell differentiation was followed by using fluorescent anti gp 135, a marker of apical membrane of polarized collecting duct cells, and visualized by confocal fluorescent microscopy. The results demonstrate that increase of GSL biosynthesis is an early event induced by hyperosmolarity which preceded the formation of apical membrane domain reflected by the apical accumulation of fluorescent gp 135. The pretreatment of cell with PDMP, a potent inhibitor of glycosyltransferase, evoked a 70% decrease of GSL biosynthesis and induced the disappearing of gp 135 accumulation in apical membrane domain. From these results, we conclude that GSL biosynthesis play a central role in the process of collecting duct cell differentiation. doi:10.1016/j.chemphyslip.2007.06.153
mutations render D-bifunctional protein deficiency as a protein folding disease. Latest results on these experiments will be discussed. Reference Ferdinandusse, Ylianttila, Gloerich, Koski, Oostheim, Waterham, Hiltunen, Wanders, Glumoff, 2006. Am. J. Hum. Genet. 78, 112–124.
doi:10.1016/j.chemphyslip.2007.06.149 PO 100
S67
imply that PAF is a putative intracellular mediator of IL-1 actions on mesangial cells and the inhibition of IL-1-elicited synthesis of PAF by OOPLE may confer protection to renal inflammation. Acknowledgement: The project is co-funded by the European Social Fund and National Resources. doi:10.1016/j.chemphyslip.2007.06.150 PO 101
Functional analysis of Pseudomonas ceramidase Nozomu Okino 1 , Makoto Ito 2 1 Department
IL-1 induces a rapid and transient increase of intracellular platelet activating factor in a human mesangial cell line—Modulation by an olive oil polar lipid extract
of Bioscience and Biotechnology, Kyushu University, Japan; 2 Department of Bioscience and Biotechnology, Kyushu University Bio-Architecture Center, Kyushu University, Japan
Tzortzis Nomikos, Elizabeth Fragopoulou, Eleftheria Rekliti, Haralabos Karantonis, Smaragdi Antonopoulou
Ceramidase (CDase) is an enzyme that catalyzes the hydrolysis of the N-acyl linkage of ceramide to generate fatty acid and sphingosine base. We previously reported the purification, molecular cloning and characterization of a neutral CDase from Pseudomonas aeruginosa strain AN17. Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (PlcH) in the genome of P. aeruginosa, which is a well-known pathogen for opportunistic infections. Recently, we found that the simultaneous production of PlcH and CDase was induced by several lipids, and PlcH-induced hemolysis was significantly enhanced by the action of CDase. When the strain was cultured with sphingomyelin or phosphatidylcholine, which is abundant in plasma membranes of mammalian cells, the production of both enzymes drastically increased, causing an increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of CDase but not PlcH. To further investigate the functional role of CDase, the gene expression of CDase was examined by RT-PCR. When the strain was cultured with sphingomyelin, which is the most effective lipid for the induction of CDase, the mRNA level of CDase was significantly elevated. These results may indicate that CDase, which is induced by plasma membrane lipids, enhances PlcH-induced cytotoxicity and provides new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.
Department of Science of Nutrition-Dietetics, Harokopio University, Greece Platelet activating factor (PAF) is a potent phospholipid inflammatory mediator implicated in the pathogenesis of glomerulosclerosis. IL-1 demonstrates a plethora of actions on human mesangial cells regulating the inflammatory response and matrix turnover within the mesangium. Olive oil possesses potent antiinflammatory properties and previous studies have shown that an olive oil polar lipid extract (OOPLE) is able to attenuate the formation of early atherosclerotic lesions in diet-induced hypercholesterolemic rabbits. Glomerulosclerosis and atherosclerosis share common histological and biochemical characteristics, therefore the objective of this study was to investigate the effect of IL-1 on PAF production by a human mesangial cell line (HMCL) and the modulation of this action by the OOPLE. IL-1 induced a dose- and time-dependent increase of intracellular PAF synthesis while no PAF was detected in the extracellular medium. The IL-1 (1ng/ml)-induced intracellular PAF elevation (2–3-fold) peaked 30 min after the stimulation, partly via activation of the lyso-PAF acetyltransferase of the remodeling biosynthetic pathway and returned to basal levels in an hour. Both basal and IL-1-stimulated PAF levels were increased by the presence of pefabloc due to PAFAH inhibition. Preincubation of HMCL with OOPLE (20–2200 ng/ml) resulted in a dose dependent inhibition of IL-1-induced PAF increase while co-incubation of IL-1 with OOPLE slightly attenuated the activation of PAF synthesis. In conclusion, the above results
S68
Abstracts / Chemistry and Physics of Lipids 149S (2007) S56–S73
References Okino, N., et al., 1998. J. Biol. Chem. 273, 14368–14373. Okino, N., et al., 1999. J. Biol. Chem. 274, 36616–36622. Okino, N., Ito, M., 2007. J. Biol. Chem. 282, 6021–6030.
it may play an important role in the control of human serum lipid/lipoprotein balance. doi:10.1016/j.chemphyslip.2007.06.152 PO 103
doi:10.1016/j.chemphyslip.2007.06.151 PO 102
OSBP-related protein 10 (ORP10), a novel regulator of lipid homeostasis Julia Perttil¨a, Krista Merikanto, Jussi Naukkarinen, Nicolas Martin, Vesa M. Olkkonen, Leena Peltonen Department of Molecular Medicine, National Public Health Institute, Finland The oxysterol-binding-protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans and are implicated in a number of vital biological functions. The role of different ORP genes and corresponding proteins in human lipid metabolism has so far not been addressed. We used genetics to study the potential role of ORP genes in regulation of human serum lipids. We tested linkage of serum lipid levels to three ORP genes (ORP2, ORP9 and ORP10) by genotyping 25 HapMap SNPs tagging their allelic diversity in 92 Finnish dyslipidemia families (ascertained for low-HDL or FCHL) with a total of 1070 individuals with well characterized serum lipid profiles. Only one of the tested ORP genes, ORP10, provided evidence for linkage both when using 5% extreme lipid levels as affected in the dichotomized linkage analysis and in the quantitative analysis using MERLIN software. Multiple SNPs of the ORP10 gene revealed evidence for linkage to the HDL and triglyceride levels (high triglycerides LODmax = 2.405 with SNP rs9853939, quantitative linkage to HDL levels LOD = 2.01, P = 0.0012 with SNP rs6807471). These results imply that allelic variants of the gene encoding ORP10 may contribute to triglyceride and HDL levels in Finnish dyslipidemic families. To obtain functional evidence for this hypothesis, we studied the effects of ORP10 silencing by short interfering siRNA on cellular lipid biosyntheses in the Huh7 (human hepatoma) cells. Knock-down of ORP10 caused a significant increase in the synthesis of cholesterol and triglycerides. Analysis by real-time RTPCR quantification revealed that ORP10 knock-down leads to an increase in the mRNA for SREBP-2, a major transcriptional regulator of cellular lipid homeostasis, suggesting that ORP10 impacts on lipids at the level of gene expression. These results identify ORP10 as a novel modulator of cellular lipid homeostasis and suggest that
Participation of the glycosphingolipid biosynthesis in the differentiation of renal collecting duct cells Lucila Pescio, Francisco Leocata-Nieto, Nicol´as Favale, Mar´ıa del Carmen Fernandez-Tom´e, Norma Sterin-Speziale Department of Biological Sciences, University of Buenos Aires, Argentina MDCK is a non-transformed renal collecting cellular line that conserves the capacity to differentiate in particular conditions of cell culture. To differentiate, collecting duct cells have to acquire a polarized phenotype. Cell polarization is characterized by the presence of apical and basolateral membrane domains which differ in their protein content as well as in lipid composition. While glycosphingolipids (GSL) predominate in the apical membrane, sphingomyelin is the major sphingolipid of the basolateral membrane. It has been previously reported that extracellular hypertonicity induces cell polarization/differentiation, but the mechanism involved is not well understood. In this study, we have examined the role of the GSL biosynthesis in the process of collecting duct cell differentiation. Confluent MDCK cells were submitted to high NaCl concentration (250 mM) for 24 and 48 h. Glycosphingolipid metabolism was determined by using 14 C-galactose as radioactive precursor. The cell differentiation was followed by using fluorescent anti gp 135, a marker of apical membrane of polarized collecting duct cells, and visualized by confocal fluorescent microscopy. The results demonstrate that increase of GSL biosynthesis is an early event induced by hyperosmolarity which preceded the formation of apical membrane domain reflected by the apical accumulation of fluorescent gp 135. The pretreatment of cell with PDMP, a potent inhibitor of glycosyltransferase, evoked a 70% decrease of GSL biosynthesis and induced the disappearing of gp 135 accumulation in apical membrane domain. From these results, we conclude that GSL biosynthesis play a central role in the process of collecting duct cell differentiation. doi:10.1016/j.chemphyslip.2007.06.153