|
|
||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045
Published, JLR Papers in Press, May 1, 2005. DOI 10.1194/jlr.M500060-JLR200
1 To whom correspondence should be addressed. e-mail: dobrowsky{at}ku.edu
| ABSTRACT |
|---|
|
|
|---|
These results suggest that the ceramide-induced loss of cholesterol from CEMs may contribute to altering the lipid raft proteome.
Abbreviations: bSMase, bacterial sphingomyelinase; Cav-1, caveolin-1; CEM, caveolin-enriched membrane; dFCS, dialyzed fetal calf serum; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MßCD, methyl-ß-cyclodextrin; SC, Schwann cell; SILAC, stable isotope labeling with amino acids in cell culture
Supplementary key words caveolae proteomics sphingolipid Schwann cells stable isotope labeling with amino acids in cell culture SILAC
| INTRODUCTION |
|---|
|
|
|---|
The integrity of lipid rafts is very dependent upon the presence of cholesterol (5). Relative to phospholipids, sphingolipids are more hydrophobic, undergo more hydrogen bonding, and tend to cluster within cell membranes (6). Moreover, the ability of cholesterol to pack tightly with saturated lipids is essential to promoting the liquid-ordered structure of lipid raft domains (6). The loss of cholesterol from caveolae by treatment with cholesterol-sequestering agents, through increased sterol oxidation, or by inhibiting its de novo synthesis leads to a more disordered state and the loss of raft-associated proteins. Indeed, because Cav-1 is a cholesterol binding protein, it is often used to monitor the loss of caveolar integrity after cholesterol displacement or depletion. Recent reports have provided evidence that ceramide production in raft model membranes and astrocytes causes a significant displacement of cholesterol from lipid raft membranes (7, 8). However, whether a ceramide-induced displacement of cholesterol can lead to changes in the protein composition of caveolin-enriched membranes (CEMs) is unknown.
The recent development of stable isotope labeling of cells in culture (SILAC) (9) has enabled a quantitative, hypothesis-driven assessment of changes in a single protein (10) as well as the unbiased, quantitative characterization of large proteomes (11). The basic premise of SILAC is that two populations of cells are grown in medium containing either unlabeled amino acids or an amino acid containing deuterium or 13C substitutions. After a sufficient incubation period to completely incorporate the labeled amino acid into the cellular proteome, one population of the cells serves as a control and the other is subjected to treatment. Cell lysates are then prepared and mixed together in a 1:1 mass ratio based upon total protein concentration. The mixed lysates may then be subjected to an enrichment procedure (immunoprecipitation, organelle isolation) for a targeted analysis, or the entire lysate can be fractionated by electrophoresis before matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or nanoelectrospray ionization MS/MS. Because minimal differences exist in the ionization efficiency between the same peptide containing a labeled versus an unlabeled amino acid, the ratio of intensities of the labeled to the unlabeled tryptic peptide provides a quantitative assessment of changes in the relative abundance of the parental protein after experimental treatment.
In this study, immortalized Schwann cells (SCs) were labeled with L-arginine ([12C]Arg) or [U-13C6]L-arginine ([13C6]Arg) and treated with bacterial sphingomyelinase (bSMase) to create ceramide-enriched lipid raft domains, and the cholesterol content and protein composition of these membranes were determined. Ceramide production markedly decreased the cholesterol content of CEMs, which was redistributed to noncaveolar membranes. Ceramide-induced cholesterol displacement was associated with a 25% decrease in the level of Cav-1, which was reversed by cholesterol replenishment. In contrast to Cav-1, bSMase treatment increased the association of 5'-nucleotidase with the CEMs but had little effect on other abundant lipid raft-associated proteins, such as flotillins and G-proteins. These results suggest that a ceramide-induced decrease in cholesterol may contribute to altering the protein composition of lipid rafts.
| EXPERIMENTAL PROCEDURES |
|---|
|
|
|---|
All cells were cultured in low-glucose DMEM that was custom prepared in 4 liter batches and conformed to Gibco DMEM number 12320, with the exception that L-arginine and L-leucine were omitted. The amino acid-deficient medium contained antibiotics and 10% dialyzed fetal calf serum (dFCS; Atlas Biologicals, Fort Collins, CO), and the addition of the necessary amino acids produced complete medium. In experiments not requiring stable isotopes, the medium was supplemented with L-leucine (d0-Leu), and [12C]Arg by the addition of 100x stock solutions prepared in serum-free leucine,arginine-deficient DMEM. In experiments requiring the stable isotopes, the medium was supplemented with L-5,5,5-trideutero-leucine (d3-Leu; Sigma/Aldrich, St. Louis, MO) or [13C6]Arg (Cambridge Isotopes, Andover, MA) by the addition of 100x stock solutions prepared in serum-free leucine, arginine-deficient DMEM.
Isolation of CEMs
Cells were placed on ice, washed with ice-cold PBS, and scraped into ice-cold MBST buffer (50 mM MES, pH 6.5, 400 mM NaCl, 1% Triton X-100, and 1x Complete® Protease Inhibitors; Roche Diagnostics). The cells were homogenized with a tight-fitting Dounce homogenizer, and CEMs were isolated by discontinuous sucrose gradient centrifugation as described previously (14). The samples were centrifuged in a SW41 rotor (126,000 g) or an MLS-50 rotor (129,000 g) for 1618 h at 4°C. For biochemical analysis of protein distribution, 10 x 0.5 ml fractions were collected from the top of the gradients. After SDS-PAGE, the proteins were transferred to nitrocellulose and the membrane was stained with 0.5% Ponceau S in 5% trichloroacetic acid to visualize total protein. For immunoblot analysis, the membrane was typically cut in half and the upper half of the blot was used to probe for flotillin-1 (BD Signal Transduction Laboratories, Lexington, KY). The lower half of the blot was used to probe for Cav-1 (N-20 antibody; Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were visualized using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Sphingomyelin, ceramide, and cholesterol measurements
Cellular sphingomyelin pools were labeled with 0.5 µCi/ml [3H]choline (American Radiolabeled Chemicals, St. Louis, MO) for 3 days. The medium was removed, and the cells were washed with ice-cold PBS and placed in serum-free DMEM for 2 h before treatment with 100 mU/ml Staphylococcus aureus bSMase (Sigma/Aldrich) for 28 h. These short incubations with bSMase did not induce any substantial decrease in cell number or increase in trypan blue permeability (data not shown). CEMs were prepared as described above, and the lipids were extracted from aliquots of the gradient fractions (15). The extent of sphingomyelin hydrolysis and ceramide production was quantified by thin-layer chromatography and the diacylglycerol kinase assay, respectively (16).
To assess the effect of ceramide production on cholesterol levels, aliquots of the individual gradient factions were used directly in the Amplex Red cholesterol assay as described by the manufacturer (Molecular Probes, Eugene, OR); the Amplex Red cholesterol assay has a linear detection range of 0.0510 µM cholesterol. To replenish cholesterol levels of the CEMs, the cells were treated for 1 h with a cholesterol-methyl-ß-cyclodextrin (MßCD) complex (17). Six milligrams of cholesterol (Avanti%20Polar%20Lipids">Avanti Polar Lipids, Alabaster, AL) was resuspended in 0.08 ml of isopropanol-chloroform (2:1), and the lipid solution was added dropwise to 200 mg of MßCD in 2.2 ml of PBS at 80°C with stirring. The resulting clear solution was diluted into cell culture medium 100-fold, giving a final cholesterol concentration of
70 µM. Preliminary experiments indicated that 1 h of incubation with the cholesterol-MßCD complex was sufficient to replenish caveolar cholesterol to at least control levels. In some experiments, cells were treated with 5 mM MßCD for 2 h to deplete caveolar cholesterol.
Quantitative proteomic analysis of CEMs by MALDI-TOF MS
S16 cells were cultured in medium containing either [12C]Arg or [13C6]Arg (810 100 mm dishes each) and served as the control and experimental populations, respectively. The cells were treated with PBS or bSMase for 28 h as described above and scraped into ice-cold MBST. The protein concentration was measured in quadruplicate for each sample using the Coomassie blue binding assay and BSA as the standard (Bio-Rad, Hercules, CA). If necessary, protein measurements were repeated so that the coefficient of variation for the protein measurements was <5% for each sample. The lysates were then mixed in a 1:1 mass ratio and typically yielded 1012 mg of total protein. CEMs were prepared, and the visible material concentrated at the interface of the 5% and 35% sucrose layers was collected. The protein was diluted with 20 volumes of 50 mM MES, pH 6.5, 400 mM NaCl, and the membranes were collected by centrifugation at 100,000 g for 1 h at 4°C. The proteins of the isolated CEMs were then resolved by SDS-PAGE.
After SDS-PAGE, the gel was stained with colloidal Coomassie blue, and the bands were excised, chopped into small pieces, and placed in a silanized 0.6 ml microcentrifuge tube. The proteins were reduced and alkylated essentially as described to prepare for in-gel digestion (18). The dehydrated gel pieces were rehydrated on ice in a minimal volume of 12.5 ng/µl Trypsin Gold (Promega Corp., Madison, WI) in 25 mM ammonium bicarbonate, pH 7.5. A sufficient volume of 25 mM ammonium bicarbonate, pH 7.5, was added to cover the gel particles, and the proteins were digested overnight at 37°C. The supernatant was transferred to a fresh tube, and the peptides were extracted from the gel particles with 5% formic acid in 100 mM ammonium bicarbonate. The combined supernatants were desalted with C-18 Zip-tips (Millipore Corp., Billerica, MA) and eluted onto the MALDI sample plate with 10 mg/ml
-cyano-carboxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid. Acetonitrile, formic acid, and ammonium bicarbonate were of the highest grade obtainable and were purchased from Fisher Scientific (Pittsburgh, PA).
Samples were analyzed by MALDI-TOF MS using an Applied Biosystems Voyager-DE STR or a 4700 Proteomics Analyzer MALDI-TOF/TOF MS/MS instrument. The instruments were operated in the positive reflector mode at the following parameters: accelerating voltage, 20,000 V; grid voltage, 75%; mirror voltage ratio, 1:12; guide wire, 0.002%; extraction delay time, 180 ns. Acquisition mass range was 7003,000 Da, and internal mass calibration was performed using the trypsin autolysis peaks (MH+ 842.5021 and 2,211.0968). Protein identification was achieved after MALDI-TOF MS or MALDI-TOF/TOF MS/MS analysis by searching the mass data against the Swiss-Prot human and rodent databases using the Mascot search engine. For protein identification by peptide mass fingerprinting, search parameters used a peptide mass tolerance of 50 ppm, up to one missed cleavage, and carboxyamidomethylated cysteine as a fixed modification. Variable modifications were set to consider methionine oxidation and the presence of [13C6]Arg. Identification was considered positive when the molecular weight search (MOWSE) score indicated that the probability of identification was P < 0.05. All spectra were further verified by manual inspection. For peptide quantification, the peak height ratios or peak area ratios were calculated using values obtained from Applied Biosystems Data Explorer software (version 4.5). Similar results were obtained using either set of values.
| RESULTS |
|---|
|
|
|---|
|
|
|
To validate that the primary SCs readily incorporated a heavy amino acid, we used d3-Leu because it is economical and is a rather abundant amino acid in most proteins. Primary SCs were grown in DMEM-10% dFCS containing d0-Leu or d3-Leu for 14 days, the cells were scraped into lysis buffer, and the protein lysates were mixed in a 1:1 mass ratio. After SDS-PAGE and staining of the gel, two abundant bands [identified as vimentin (52 kDa) and ß-actin (42 kDa)] were excised and subjected to in-gel reduction, alkylation, and tryptic digestion. A strong benefit of SILAC is that the rapid identification of d3-Leu-containing peptides is facilitated by the presence of a characteristic doublet of peak clusters that differ in mass by the number of leucine residues in the peptide and multiplied by 3. Figure 3A shows a representative peak doublet for the labeled and unlabeled 171LQEEMLQR178 peptides of vimentin. The presence of two leucine residues in the unlabeled peptide (m/z 1,046.39) results in a shift in the mass of the d3-Leu-containing peptide by 6 Da (m/z 1,052.41). The peaks at m/z 1,047.39 and 1,048.39 (1,053.41 and 1,054.41 in the d3-Leu peptide) form the isotope cluster of the peptide. These peaks arise from the 1% probability that the natural abundance of the 13C isotope replaces any 12C atom in the unlabeled peptide (21). We verified that these doublets represent actual d0-Leu and d3-Leu peak clusters by MALDI-TOF/TOF MS/MS. The precursor mass window of ±1.0 Da was centered on the most abundant isotopomers of the peptide doublet, and MS/MS fragmentation of the peptide bonds produced an extensive series of b-ions and y-ions of the unlabeled (Fig. 3B) and deuterated (Fig. 3C) peptides. The resulting peptide sequence tags provided definitive identification of this vimentin peptide and the location of the d3-Leu residues. For example, leucine is at the N terminus of this peptide. This is supported by the entire series of b-ions (b1 to b5) that were shifted by 3 Da in the deuterated species and by the presence of a1-ions at m/z 86.11 and 89.13 for d0-Leu and d3-Leu, respectively. Similarly, the observed y4- and y5-ions (but not the y1- and y2-ions) also showed a shift of 3 Da, consistent with the presence of d3-Leu as the sixth amino acid.
|
4550%. Importantly, any increase in the intensity of the isotopomer at m/z 1,257.96 would indicate the presence of a monodeuterated species. However, the abundance of this peak did not increase relative to that present in unlabeled cells (day 0). This indicates that d3-Leu has effectively saturated the leucine-tRNA pool and that d3-Leu present in the medium is the primary source of this amino acid for new protein synthesis. By day 8, the unlabeled peptide accounted for <10% of the mass, and it was essentially lost after 10 days of incubation in medium containing d3-Leu. Similar results were obtained when lysates from cells grown in either d0-Leu or d3-Leu for various times were mixed on an equal mass basis. In this approach, complete incorporation of the d3-Leu was also evident by 10 days, because the ratio of unlabeled to labeled peptide reaches unity (Fig. 5A).
|
|
Relative protein quantification by SILAC
To establish some analytical limits for quantitation by SILAC using MALDI-TOF MS, we measured changes in known mixtures of unlabeled and labeled proteins that varied over a range of 25400%. Cells were grown in d0-Leu or d3-Leu for 10 days, and the lysates were mixed to give various theoretical mass ratios of d0-Leu to d3-Leu (1:0.5, 1:0.75, 1:1, 1:1.33, 1:2, and 1:4). An aliquot of each mixture was subjected to SDS-PAGE, and the vimentin bands were analyzed by MALDI-TOF MS. The observed ratios of nondeuterated-to-deuterated peptide were typically within 10% of the theoretical ratios, with the exception of the standard mixture at a ratio of 1:1.33 (Fig. 6A). However, upon averaging of the observed peak height ratios of the deuterated-to-nondeuterated peptides over six doublets that contained one, two, or four leucine residues in each of the six mixtures, there was very good agreement with the expected theoretical ratios (Fig. 6B). Although the linearity of the response began to deviate in the quantitation of greater fold differences, this was not attributable to signal saturation in the mass spectrometer. These data suggest that we may underestimate peptides changing by 4-fold or greater. Although we have not directly identified the source of this error, it may arise at least in part from a greater percentage contribution to the signal intensity from background noise in the unlabeled sample.
|
Effect of bSMase on the protein profile of CEMs from S16 cells
S16 cells are an immortalized line of rat SCs that undergo rapid population doubling compared with primary SCs. Although the S16 cells readily incorporated [13C6]Arg to a sufficient level within 5 days (data not shown), they were maintained upon subculturing in medium supplemented with [13C6]Arg for 2030 passages. Under these conditions, the level of incorporation of the label should theoretically exceed 99.99%. S16 cells were treated with buffer ([12C]Arg) or 100 mU/ml bSMase ([13C6]Arg) for 28 h, and cell lysates were prepared. After mixing the lysates in a 1:1 mass ratio, CEMs were isolated and the proteins were resolved by SDS-PAGE and prepared for analysis by MALDI-TOF MS.
Cav-1 is a cholesterol binding protein (23) whose association with CEMs is decreased upon depleting cellular cholesterol with cholesterol binding agents such as MßCD (24). Figure 7A shows a representative peptide mass fingerprint for rat Cav-1. Evident is the presence of five [12C]Arg:[13C6]Arg peptide doublets that aided both quantification and identification. Indeed, Table 1 shows that, on average, five [12C]Arg:[13C6]Arg tryptic peptide doublets were used for quantification versus 910 total tryptic peptides ([12C]Arg- + [13C6]Arg- + [12C]lysine-containing peptides) that facilitated identification.
|
|
25% (Fig. 8A). Importantly, replenishing cholesterol for 1 h, after 7 h of treatment with bSMase, reversed the loss of Cav-1 (Fig. 8B). These results support the notion that the loss of Cav-1 from the CEMs was associated with the ceramide-induced cholesterol depletion. In contrast, bSMase treatment did not substantially alter the level of G-proteins, flotillin-1, and flotillin-2 (Table 2, Fig. 8A), which undergo a substantial decrease in lipid rafts after cholesterol depletion by MßCD (25). Indeed, the G-protein, Gi-
3, was substantially depleted from the CEMs after 2 h of treatment with 5 mM MßCD but was essentially unchanged by bSMase treatment (Fig. 9). On the other hand, prolonged incubation with bSMase induced a 50% increase in 5'-nucleotidase, a well-established raft protein (25), and an
50% decrease in the recently identified raft protein ATP synthase-ß chain (26).
|
|
| DISCUSSION |
|---|
|
|
|---|
At this point, our study does not address how ceramide generation may displace cholesterol from lipid raft domains. The interaction of cholesterol with sphingomyelin occurs through hydrogen bonding of the C3-hydroxyl of cholesterol with the sphingosine backbone of sphingomyelin. Although this structure is maintained in ceramide, the loss of the polar phosphocholine group results in ceramide having a very poor affinity for cholesterol. Indeed, biophysical studies have shown that ceramides mix very poorly with cholesterol and spontaneously undergo lateral phase separation and the formation of a ceramide-enriched microdomain (29). Additionally, recent evidence also indicates that ceramide phosphoethanolamine, which lacks the three methyl groups of the phosphocholine head group present in sphingomyelin, does not interact favorably with cholesterol (30). These data strongly support the notion that the phosphocholine head group is also important for stabilizing sphingolipid-sterol interactions (30). Thus, although ceramides generated from sphingomyelin hydrolysis share the same sphingoid base and fatty acid composition, this is not sufficient to promote a cholesterol-ceramide interaction. Conceivably, as sphingomyelin is hydrolyzed, the production of ceramide-enriched microdomains creates a poor environment for cholesterol-sphingolipid interactions. Consistent with this possibility, SMase D was substantially less effective than SMase C at mobilizing free cholesterol, despite decreasing sphingomyelin levels to a similar extent (28). Because SMase D hydrolyzes sphingomyelin to ceramide-1-phosphate, this phosphorylated ceramide derivative does not mimic the actions of ceramide in the raft domains. These results support the idea that cholesterol displacement is associated with increased ceramide production rather than the loss of sphingomyelin per se (28).
To facilitate characterizing the effect of ceramide-induced cholesterol depletion on the protein composition of the CEMs, we used a stable isotope labeling procedure that permits a quantitative proteomic analysis between two cell populations. SILAC has been used to provide both targeted (10, 31) and more global quantitative analyses of cellular proteomes (20, 25). We have documented the suitability of this approach using both primary SCs and immortalized S16 SCs. Primary SCs readily incorporated d3-Leu into the cellular proteome with essentially no effect on cell growth and viability. We have also used [13C6]Arg and [13C9]tyrosine to label primary SCs and have encountered no problems with incorporation of the labeled amino acids into the proteome (data not shown). As a secondary note, some cell lines can catabolize arginine to proline, which would produce [13C5]proline in cells supplemented with [13C6]Arg (32). Although this is not necessarily problematic for quantitation by SILAC, we observed no conversion of [13C6]Arg to [13C5]proline in either primary SCs or the S16 immortalized SCs.
The lower analytic limits for quantitation by SILAC were found to be an
25% change in protein abundance. However, it should be noted that accurate quantitation of changes of this magnitude is facilitated by a high signal-to-noise ratio and is strengthened by quantifying as many peptide doublets as possible. Although the use of both [13C6]Arg and [13C6]lysine would further increase the number of peptide doublets available for quantitation (33), it has been our experience that lysine-containing peptides are poorly represented in the MALDI-TOF spectra, as has been reported previously (34). Collectively, these results provide the first evidence that SILAC analysis is feasible using either primary or immortalized SCs.
Overall, our MALDI-TOF analysis reproducibly identified and quantified 10 bona-fide caveolar proteins over the course of several different cell preparations and treatments (Table 2). Most of these proteins have been identified previously as lipid raft proteins based upon their 7.5- to10-fold decrease in association with CEMs after a 95% depletion of cholesterol using MßCD (25). Surprisingly, this level of cholesterol depletion resulted in only a 3-fold decrease in Cav-1 (25). Although bSMase treatment decreased cholesterol levels by 2550% within 2 h, the relative abundance of Cav-1 decreased by 25% only after 8 h. On the other hand, ceramide-induced cholesterol depletion correlated with greater changes in the relative abundance of the ß-subunit of ATP synthase and 5'-nucleotidase but had little effect on altering the association of flotillins or G-proteins with CEMs. Overall, it was quite surprising that a loss of 2550% of the cholesterol content of the CEMs did not generate greater changes in the protein content of the membranes. Because ceramide-rich domains form highly ordered physical states in membranes and compete with cholesterol for association with rafts (7), it is possible that ceramide may substitute, in part, for the loss of cholesterol and protect some proteins from exiting the CEMs. The ability of Cav-1 to bind cholesterol may render this protein slightly more susceptible to exiting from the CEMs as ceramide displaces cholesterol. To examine this possibility further, we attempted to identify the receptor for the hedgehog proteins, Patched. Patched is expressed in SCs (35), is responsive to changes in cholesterol levels, binds to Cav-1, and localizes to lipid rafts (36). However, the identification of the rat homolog of Patched from the mouse or human protein sequences was not of high enough probability to allow a confident identification.
Finally, although bSMase is a pharmacological tool for increasing ceramide, the magnitude of the increase in ceramide is well within a range that is often produced by agonists or cell stress. Our observed loss of cholesterol from the CEMs, and its effect on the abundant structural proteins, may therefore be representative of physiological changes in these domains after the activation of plasma membrane SMases. However, the lipid and protein composition of lipid rafts is not necessarily identical between different cell types, and this may influence the rate of cholesterol and/or protein displacement induced by ceramide generation. Additionally, one must also consider that agonists also activate other sphingolipid-metabolizing enzymes (37) that may contribute to regulating the ceramide content and protein composition of lipid rafts.
In summary, our work suggests that both ceramide production and the loss of cholesterol from lipid rafts contribute to changes in the protein composition of these specialized membrane microdomains. Additionally, we demonstrate the feasibility of using SILAC in both immortalized and primary SCs to provide a quantitative proteomic approach to investigate signal transduction events in these cells. The application of SILAC analysis in the identification and quantification of changes in both protein abundance and protein phosphorylation (38) provides a useful tool for characterizing the effect of ceramide signaling on cellular or organellar proteomes.
| ACKNOWLEDGMENTS |
|---|
Manuscript received February 16, 2005 and in revised form April 12, 2005.
| REFERENCES |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
F. Galbiati, V. Basso, L. Cantuti, M. I. Givogri, A. Lopez-Rosas, N. Perez, C. Vasu, H. Cao, R. van Breemen, A. Mondino, et al. Autonomic Denervation of Lymphoid Organs Leads to Epigenetic Immune Atrophy in a Mouse Model of Krabbe Disease J. Neurosci., December 12, 2007; 27(50): 13730 - 13738. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Rebillard, X. Tekpli, O. Meurette, O. Sergent, G. LeMoigne-Muller, L. Vernhet, M. Gorria, M. Chevanne, M. Christmann, B. Kaina, et al. Cisplatin-Induced Apoptosis Involves Membrane Fluidification via Inhibition of NHE1 in Human Colon Cancer Cells Cancer Res., August 15, 2007; 67(16): 7865 - 7874. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. E. Furland, G. M. Oresti, S. S. Antollini, A. Venturino, E. N. Maldonado, and M. I. Aveldano Very Long-chain Polyunsaturated Fatty Acids Are the Major Acyl Groups of Sphingomyelins and Ceramides in the Head of Mammalian Spermatozoa J. Biol. Chem., June 22, 2007; 282(25): 18151 - 18161. [Abstract] [Full Text] [PDF] |
||||
![]() |
K. Krishnamurthy, S. Dasgupta, and E. Bieberich Development and characterization of a novel anti-ceramide antibody J. Lipid Res., April 1, 2007; 48(4): 968 - 975. [Abstract] [Full Text] [PDF] |
||||