Journal: Nutrients

Article Title: Chronic Oleoylethanolamide Treatment Decreases Hepatic Triacylglycerol Level in Rat Liver by a PPARγ/SREBP-Mediated Suppression of Fatty Acid and Triacylglycerol Synthesis

doi: 10.3390/nu13020394

Figure Lengend Snippet: Hepatic sphingolipid and neutral lipid analysis. Total lipids from VEH and OEA groups were extracted and separated by TLC. ( a ) Representative TLC separation of sphingolipids. ( b , c ) Quantification of ceramide and sphingomyelin by densitometry analysis. Ceramide and sphingomyelin level is reported as fold change of OEA versus VHE. ( d ) Neutral and acid sphingomyelinase and sphingomyelin synthase activities reported as percentages of values measured in VEH. ( e ) Ceramidase activity assayed in liver homogenates. ( f ) Representative TLC separation of neutral lipids. ( g ) Neutral lipids are represented as percentage of the total. ( h ) Triacylglycerol (TAG) amount was spectrophotometrically quantified with a specific enzymatic assay. In the figure, the mean ± SEM of values obtained from three different analyses is reported. ** p < 0.005; * p < 0.05. Abbreviations: Chol. Est. = cholesterol ester; TAG = triacylglycerols: FFA = free fatty acids; DAG = diacylglycerols.

Article Snippet: The enzymatic assay was conducted with a Colorimetric Sphingomyelinase Assay Kit furnished by MERK (Italy).

Techniques: Activity Assay, Enzymatic Assay

Journal: Journal of Biological Chemistry

Article Title: Transient Mechanoactivation of Neutral Sphingomyelinase in Caveolae to Generate Ceramide

doi: 10.1074/jbc.m210375200

Figure Lengend Snippet: FIG. 1. Acute mechanoactivation of N- but not A-SMase in en- dothelial cell plasma membranes. Rat lung vasculature was per- fused in situ first for 5 min at 6–8 mm Hg and then at a higher pressure of 14–15 mm Hg for the time indicated. The activity of either N-SMase (filled symbols) or A-SMase (open symbols) in the rat lung homogenates (H, squares) or the isolated luminal endothelial cell plasma membranes (P, circles) was measured as described under “Experimental Proce- dures.” The percentage of the enzyme activity compared with time zero is presented. Data are the average of two experiments (n 2).

Article Snippet: Materials—Reagents and other supplies were obtained from the following sources: [N-methyl-14C]sphingomyelin (55 mCi/mmol) and Percoll from Amersham Biosciences; imidazole, ceramide, DETAPAC, octyl- -Dglucopyranoside, sphingomyelinase (Staphylococcus aureus), glycerophosphate, anti-diphosphorylated ERK1 and 2 monoclonal antibody, and antipan ERK antibody from Sigma; cardiolipin from Avanti Polar Lipids (Alabaster, AL); diacylglycerol kinase from Calbiochem (San Diego, CA); [ -32P]ATP (3000 Ci/mmol) from PerkinElmer Life Sciences; the bicinchoninic acid (BCA) protein assay kit from Pierce; anti-acid sphingomyelinase (A-SMase) antibodies from Santa Cruz Biotechnology; anti-caveolin-1 monoclonal antibody (2234) from BD Biosciences; M-450 Dynabeads from Dynal (New Hyde Park, NY); and Optiprep from Invitrogen.

Techniques: Clinical Proteomics, In Situ, Activity Assay, Isolation

Journal: Journal of Biological Chemistry

Article Title: Transient Mechanoactivation of Neutral Sphingomyelinase in Caveolae to Generate Ceramide

doi: 10.1074/jbc.m210375200

Figure Lengend Snippet: FIG. 2. Mechanoactivation of N-SMase and subsequent ceramide formation. A and B, rat lung vasculature perfusion was maintained either under control conditions (6–8 mm Hg, open circles) or elevated to high pressure/flow conditions (14–15 mmHg, filled circles) for the time indicated. C and D, the perfusions were continued for 2 min at pressures ranging from 6–18 mm Hg to give the indicated flow rates. The luminal endothelial plasma membranes were isolated and used to measure N-SMase activity (panels A and C) and ceramide levels (panels B and D). Data are expressed as the mean S.D. (n 4).

Article Snippet: Materials—Reagents and other supplies were obtained from the following sources: [N-methyl-14C]sphingomyelin (55 mCi/mmol) and Percoll from Amersham Biosciences; imidazole, ceramide, DETAPAC, octyl- -Dglucopyranoside, sphingomyelinase (Staphylococcus aureus), glycerophosphate, anti-diphosphorylated ERK1 and 2 monoclonal antibody, and antipan ERK antibody from Sigma; cardiolipin from Avanti Polar Lipids (Alabaster, AL); diacylglycerol kinase from Calbiochem (San Diego, CA); [ -32P]ATP (3000 Ci/mmol) from PerkinElmer Life Sciences; the bicinchoninic acid (BCA) protein assay kit from Pierce; anti-acid sphingomyelinase (A-SMase) antibodies from Santa Cruz Biotechnology; anti-caveolin-1 monoclonal antibody (2234) from BD Biosciences; M-450 Dynabeads from Dynal (New Hyde Park, NY); and Optiprep from Invitrogen.

Techniques: Control, Clinical Proteomics, Isolation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Transient Mechanoactivation of Neutral Sphingomyelinase in Caveolae to Generate Ceramide

doi: 10.1074/jbc.m210375200

Figure Lengend Snippet: FIG. 3. Enrichment of N- but not A-SMase in the endothelial luminal surface plasma membranes and their caveolae. The ac- tivity of N-SMase (panels A and B) or A-SMase (panel C) was measured in 10 g of rat lung homogenate (H), purified silica-coated luminal endothelial cell plasma membranes (P), caveolae isolated either in the presence (V in panels A and C) or absence (V in panel B) of Triton X-100, the Triton-soluble phase (T), the 40% sucrose fraction (S, in the absence of detergent), or resedimented silica-coated plasma membranes stripped of caveolae (P-V in panels A and C or P-V in panel B). Data are expressed as the mean S.D. (n 3) (panel B) and the mean from two experiments (panels A and C). D, Western analysis of the indicated subfractions using antibodies specific for caveolin, 5-NT, and -actin.

Article Snippet: Materials—Reagents and other supplies were obtained from the following sources: [N-methyl-14C]sphingomyelin (55 mCi/mmol) and Percoll from Amersham Biosciences; imidazole, ceramide, DETAPAC, octyl- -Dglucopyranoside, sphingomyelinase (Staphylococcus aureus), glycerophosphate, anti-diphosphorylated ERK1 and 2 monoclonal antibody, and antipan ERK antibody from Sigma; cardiolipin from Avanti Polar Lipids (Alabaster, AL); diacylglycerol kinase from Calbiochem (San Diego, CA); [ -32P]ATP (3000 Ci/mmol) from PerkinElmer Life Sciences; the bicinchoninic acid (BCA) protein assay kit from Pierce; anti-acid sphingomyelinase (A-SMase) antibodies from Santa Cruz Biotechnology; anti-caveolin-1 monoclonal antibody (2234) from BD Biosciences; M-450 Dynabeads from Dynal (New Hyde Park, NY); and Optiprep from Invitrogen.

Techniques: Clinical Proteomics, Purification, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transient Mechanoactivation of Neutral Sphingomyelinase in Caveolae to Generate Ceramide

doi: 10.1074/jbc.m210375200

Figure Lengend Snippet: FIG. 4. N-SMase but not A-SMase in immuno-isolated caveolae. BLMVEC homogenates were fractionated on a Percoll gradient to isolate the PM from the post nuclear supernatant (PNS) followed by sonication of PM and sucrose gradient centrifugation to isolate AC. AC was subsequently subjected to immuno-affinity isolation using caveolin-1 antibody-conjugated magnetic beads to separate caveolae bound to the beads (B) from noncaveolar unbound material (U) in the supernatant (31). Western analysis was performed on each fraction using antibodies to the indicated proteins.

Article Snippet: Materials—Reagents and other supplies were obtained from the following sources: [N-methyl-14C]sphingomyelin (55 mCi/mmol) and Percoll from Amersham Biosciences; imidazole, ceramide, DETAPAC, octyl- -Dglucopyranoside, sphingomyelinase (Staphylococcus aureus), glycerophosphate, anti-diphosphorylated ERK1 and 2 monoclonal antibody, and antipan ERK antibody from Sigma; cardiolipin from Avanti Polar Lipids (Alabaster, AL); diacylglycerol kinase from Calbiochem (San Diego, CA); [ -32P]ATP (3000 Ci/mmol) from PerkinElmer Life Sciences; the bicinchoninic acid (BCA) protein assay kit from Pierce; anti-acid sphingomyelinase (A-SMase) antibodies from Santa Cruz Biotechnology; anti-caveolin-1 monoclonal antibody (2234) from BD Biosciences; M-450 Dynabeads from Dynal (New Hyde Park, NY); and Optiprep from Invitrogen.

Techniques: Isolation, Sonication, Gradient Centrifugation, Magnetic Beads, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transient Mechanoactivation of Neutral Sphingomyelinase in Caveolae to Generate Ceramide

doi: 10.1074/jbc.m210375200

Figure Lengend Snippet: FIG. 5. Acute mechanoactivation of N-SMase in caveolae. After perfusion first at 6–8 mm Hg for 5 min and then either at 6–8 mm Hg (open bars) or 14 mm Hg (hatched bars) for 2 min, the subcellular fractions (H, P, V, S, P-V) were obtained in the absence of detergent. Equal amounts of each subcellular fraction (10 g of protein) were measured for the activity of N-SMase. Data are expressed as the mean S.D. (n 3).

Article Snippet: Materials—Reagents and other supplies were obtained from the following sources: [N-methyl-14C]sphingomyelin (55 mCi/mmol) and Percoll from Amersham Biosciences; imidazole, ceramide, DETAPAC, octyl- -Dglucopyranoside, sphingomyelinase (Staphylococcus aureus), glycerophosphate, anti-diphosphorylated ERK1 and 2 monoclonal antibody, and antipan ERK antibody from Sigma; cardiolipin from Avanti Polar Lipids (Alabaster, AL); diacylglycerol kinase from Calbiochem (San Diego, CA); [ -32P]ATP (3000 Ci/mmol) from PerkinElmer Life Sciences; the bicinchoninic acid (BCA) protein assay kit from Pierce; anti-acid sphingomyelinase (A-SMase) antibodies from Santa Cruz Biotechnology; anti-caveolin-1 monoclonal antibody (2234) from BD Biosciences; M-450 Dynabeads from Dynal (New Hyde Park, NY); and Optiprep from Invitrogen.

Techniques: Activity Assay