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PubMed2000

Ether--lipid (alkyl-phospholipid) metabolism and the mechanism of action of ether--lipid analogues in Leishmania.

Ether-lipid (alkyl-phospholipid) analogues such as Miltefosine possess potent in vitro and in vivo anti-leishmanial activity and these compounds are currently undergoing clinical trials in humans. These analogues are also effective against Trypanosoma cruzi and Trypanosoma brucei subspecies but their mode of action is not known. Leishmania have high levels of ether-lipids and these are mainly found in the glycosylphosphatidylinositol-anchored glycolipids and glycoproteins present on the surface of the parasites. In Leishmania mexicana promastigotes we have studied both the initiating steps for the biosynthesis of ether-lipids, and key remodelling steps. The effect of Miltefosine and Edelfosine, on key enzymes involved in the metabolism of ether-lipids has been studied. The enzymes include dihydroxyacetonephosphate acyltransferase, sn-l-acyl-2-lyso-glycero-3-phosphocholine and sn-l-alkyl-2-lyso-glycero-3-phosphocholine acyltransferases. We confirm that the initiating steps in ether-lipid metabolism in Leishmania are present in glycosomes, and that Miltefosine or Edelfosine did not perturb these enzymes. The metabolism of the latter phosphatidylcholine base intermediates, which may be involved in the remodelling of acyl- and alkyl-glycerophospholipids, was also seemingly associated with glycosomes. Both Miltefosine and Edelfosine inhibited this microbody (glycosomal) located alkyl-specific-acyl-CoA acyltransferase in a dose-dependent manner with an inhibitory concentration of 50 microM. It is suggested therefore that a perturbation of ether-lipid remodelling could be responsible for the anti-leishmanial action of these drugs.

Acylation↗
PubMed2001

Magnetic resonance lipid signals in rat brain after experimental stroke correlate with neutral lipid accumulation.

Proton magnetic resonance spectroscopy (MRS) signals from lipids in brain have been observed to increase after ischemic brain injury. However, neither the chemical identity nor the cellular location of these lipids has been established. The aim of the present study was to identify the origin of MRS lipid signals in rat brain after temporary (90 min) middle cerebral artery occlusion (MCAO). Fatty acyl proton signals were detected by short-echo one and two dimensional (1)H MRS in superfused brain slices from the infarcted hemisphere 1-5 days after MCAO. The intensities of these signals were strongly correlated with the amount of triacylglyceride and cholesterol ester in lipid extracts from the samples (r(2)=0.96, P<0.05) and were not correlated with the amount of free fatty acids in the tissue. Histological staining of tissue revealed the presence of neutral lipid droplets in infarcted regions. Dual labeling by immunohistochemistry demonstrated that these droplets were localized to microglia/macrophage (OX-42-labeled cells). These results strongly suggest that (1)H MRS lipid signals from brain after stroke arise from microglia/macrophage phagocytosis of cellular membranes.

Animals↗
PubMed2000

Systematic, immediate in-hospital initiation of lipid-lowering drugs during acute coronary events improves lipid control.

Background: Patients who have had a coronary heart attack often go completely untreated for hypercholesterolemia. We investigated whether immediate initiation of lipid-lowering drugs during hospitalization for acute coronary events increases the proportion of correctly treated patients compared to referred treatment as recommended by current guidelines. Methods: This prospective, multicenter study randomized 57 hypercholesterolemic patients hospitalized for acute coronary events to immediate in-hospital initiation or to referred initiation of lipid-lowering drugs by primary care physicians 3 months after unsuccessful nutritional intervention. Results: After 6 months, 53 patients were available for follow-up. More patients in the immediate initiation group (26/30 patients, 87%) were treated with lipid-lowering drugs than in the referred initiation-group (13/23 patients, 57%, P=0.03). Twenty-seven patients (87%) in the immediate initiation group versus 17 patients (65%) in the referred initiation group had a 10% or greater decrease in total cholesterol or a 15% or greater decrease in LDL-cholesterol (P=0.18). Although statistically not significant, there was a trend to improved lipid values in the immediate initiation group compared to the referred initiation group (TC, -21.1 vs. -13.8% (P=0.08); LDL-C, -28.2 vs. -18.9% (P=0.13); HDL-C, +10.8 vs. +5% (P=0.44); TC/HDL-C ratio, -24.7 vs. -15.1% (P=0.22)), and the LDL-C/HDL-C ratio was -34.1 vs. -19.1% (P=0.04, P=NS after Bonferroni correction). Conclusion: The immediate initiation of lipid-lowering drugs in hypercholesterolemic patients hospitalized for acute coronary events increases the rate of correctly treated patients and has the potential to improve lipid control.

Journal Article↗
PubMed1994

Selective transfer of cholesteryl ester over triglyceride by human plasma lipid transfer protein between apolipoprotein-activated lipid microemulsions.

The substrate-specific rate of the human plasma lipid transfer protein (LTP) reaction was studied using pyrene-labeled substrate lipid analogues as probes for various lipids, by monitoring the ratio of the fluorescence intensities of their excimers to those of their monomers as an indicator of pyrene concentration in the microenvironment. Transfer of cholesteryl ester (CE) and triglyceride (TG) was demonstrated between human high-density lipoproteins, between low-density lipoproteins, and between these two lipoprotein, and the specific fractional transfer rate of CE was always higher than that of TG by a factor of 2.4-7.9. On the other hand, the transfer by LTP of CE, TG, and phosphatidylcholine (PC) was also demonstrated between lipid microemulsions having an average diameter of 25-26 nm using the same probes, but only when the emulsions were activated by apolipoproteins A-I, A-II, E, or C-III. The maximally activated rates of the transfer of CE and TG were the same when measured between the emulsions with cores composed exclusively of either lipid. The specific fractional transfer rate of pyrene-CE, however, was inversely proportional to the percentage of CE in the TG core of the emulsions, and the initial transfer of TG was almost completely inhibited by the presence of small percentages of CE in the TG core. Thus, the transfer of CE between the emulsions is highly selective over that of TG by orders of magnitude, much more selective than the reaction between any natural plasma lipoproteins, but this selectivity is not a rate-limiting step of the overall LTP reaction. The maximally activated LTP-catalyzed transfer rate of PC between the emulsions was somewhat higher than that of CE or TG and was not affected by the composition of the core lipids of the emulsion, TG or CE. When an excess amount of LTP was incubated with emulsion containing a small percentage of pyrene-CE in the TG core in the absence of the acceptor particles, excimer fluorescence rapidly decreased to the base line, and this change was suppressed when pyrene-CE was diluted with CE in the core. This result may indicate that LTP selectively disrupts pyrene-CE excimer formation on the basis of its selective interaction with the CE molecule over TG in the emulsion system as a putative background mechanism for the selective transfer of CE.

Apolipoprotein A-I↗
PubMed1981

Mechanism of lipid-protein interaction in the plasma lipoproteins: identification of a lipid-binding site in apolipoprotein A-II.

Apolipoprotein A-II (apoA-II) is a dimeric 77-residue apoprotein of human high-density lipoproteins. Previous studies indicate that residues 56--77 in the apoprotein do not bind phospholipid whereas residues 47--77 form a complex with dimyristoylphosphatidylcholine (DMPC). To further delineate the lipid-binding region between residues 47 and 77, we have prepared synthetic fragments of apoA-II corresponding to residues 54--77, 52--77, and 50--77 and have tested each fragment for its ability to interact with vesicles of DMPC. The interaction of the fragments was determined by changes in secondary structure as measured by circular dichroism and by isolation of peptide--DMPC complexes by ultracentrifugation in density gradients of KBr. By these criteria, only fragment 50--77 binds DMPC; there is an increase in alpha helicity from 17% to 41% when the fragment associates with lipid. Since the 56--77 fragment does not associate with phospholipid, we propose that the addition of residues Thr-Pro-Leu-Ile-Lys-Lys (corresponding to residues 50--55) to the 56--77 fragment gives the peptide the necessary sequence information for lipid binding. To further identify the important amino acid residues in the region of 50--55, we have substituted Leu-Ile with Ala-Ala. This substitution totally abolishes the lipid-binding capacity of the 50--77 fragment. On the other hand, substitution of Lys-Lys with Ser-Ser does not alter the lipid-binding capacity of the peptide. We conclude that residues 50--55 are important in lipid binding and that the hydrophobic center formed by Leu-Ile plays an important role.

Amino Acid Sequence↗
PubMed2001

Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.

The binding of two mono-acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C-terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1-myristoylglycerophosphatidylcholine (MyrGro-PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 microM and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro-PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen-addad, C. & Pebay-Peyroula, E. (1999) Eur. J. Biochem. 264, 562-568.]. Results from isothermal titration calorimetry experiments indicated non-classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two omega-hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.

Calorimetry↗
PubMed2000

The N-terminal beta-barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies.

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post-translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A beta-barrel-forming N-terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the beta-barrel represents a membrane-binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact beta-barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX beta-barrel with glutathione S-transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST-fusion protein confirmed the role of the beta-barrel as the membrane-targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX-1. When the beta-barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.

Binding Sites↗
PubMed1976

Biosynthesis of lipid-linked oligosaccharides and glycoprotein in aorta: stimulation by acceptor lipids isolated from liver.

An acidic glycolipid fraction was isolated from porcine liver and partially purified on DEAE-cellulose. This material markedly stimulated the incorporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein, but did not stimulate incorporation into the mannosyl-phosphoryl-polyprenol. The products formed from GDP-[14C]mannose in the presence of acceptor lipid were a series of lipid-linked oligosaccharides of mannose and N-acetylglucosamine (GlcNAc) which differ from each other in the size of the oligosaccharide moieties. The acceptor lipid fraction was partially characterized in terms of its carbohydrate composition by mild acid hydrolysis and reduction with NaB3H4. The [3H]sugars were then separated into a number of monosaccharides and oligosaccharides by paper chromatography. Strong acid hydrolysis of two of the larger oligosaccharides released from the acceptor lipid followed by reduction with NaB3H4 gave [3H]mannitol and [3H]GlcN-ol in ratios of 2.8:2 and 3.8:2, indicating that these compounds probably were penta- and hexasaccharides. In addition, GlcNAc was shown to be at the reducing terminus and alpha-linked mannose at the nonreducing ends of the molecules. The results suggest that the acceptor lipid fraction is composed of oligosaccharides of (Man)n leads to GlcNAc leads to GlcNAc, probably attached to a polyprenol through a pyrophosphoryl linkage.

Animals↗
PubMed1990

Hepatic biliary lipid secretion and gall bladder biliary lipid mass in gall stone patients: effect of ursodeoxycholic acid.

We have carried out overnight measurements of hepatic secretion rate and duodenal output of biliary lipids using a duodenal perfusion technique. We correlated these measurements with the fasting state mass of biliary lipids within the gall bladder on the following morning using a combined nasoduodenal intubation and isotope scanning technique. We studied six gall stone subjects before and during treatment with ursodeoxycholic acid 675 mg/day. Lipid mass within the gall bladder correlated with the corresponding overnight hepatic secretion rate for all three biliary lipids. During ursodeoxycholic acid treatment, there was an increase in gall bladder bile acid mass without significant change in cholesterol or phospholipid mass. We conclude that the mass of individual biliary lipids within the fasting gall bladder is influenced by overnight hepatic biliary lipid secretion rate; and that the effect of ursodeoxycholic acid (675 mg/day) on cholesterol saturation index of fasting gall bladder bile is mediated via an increase in bile acid mass rather than through a decrease in cholesterol mass within the gall bladder.

Aged↗
PubMed2003

CETP and lipid transfer inhibitor protein are uniquely affected by the negative charge density of the lipid and protein domains of LDL.

Lipoprotein surface charge influences cholesteryl ester transfer protein (CETP) activity and its association with lipoproteins; however, the relationship between these events is not clear. Additionally, although CETP and its regulator, lipid transfer inhibitor protein (LTIP), bind to lipoproteins, it is not known how the charge density of lipoprotein protein and lipid domains influences these factors. Here, the electronegativity of the protein (by acetylation) and surface lipid (oleate addition) domains of LDL were modified. LDL-only lipid transfer assays measured changes in CETP and LTIP activities. CETP activity was stimulated by <10 microM oleate but completely suppressed by >20 microM. The same electronegative potential induced by acetylation mildly stimulated CETP. Modification-induced enhanced binding of CETP did not correlate with CETP activity. LTIP activity was completely blocked by approximately 10 microM oleate but only mildly suppressed by acetylation. LTIP binding to LDL was not decreased by oleate. Thus, the negative charge of LDL surface lipids, but not protein, is an important regulator of CETP and LTIP activity. Altered binding could not explain changes in CETP activity, suggesting that the extent of CETP binding is not normally rate limiting to its activity. Physiologic and pathophysiologic conditions that modify the negative charge of lipoprotein surface lipids will suppress LTIP activity first, followed by CETP.

Carrier Proteins↗
PubMed1999

Effect of oxidized lipids in the diet on oxidized lipid levels in postprandial serum chylomicrons of diabetic patients.

OBJECTIVE: To determine whether humans with type 2 diabetes have increased levels of oxidized fatty acids in their serum chylomicron fraction after the ingestion of dietary oxidized fatty acids. RESEARCH DESIGN AND METHODS: The study was performed on 31 male type 2 diabetic patients and 24 age-matched control subjects. Among the diabetic patients, 22 had poor glycemic control, defined as HbA1 > 10% (normal value < 7.7%). Nine patients had good glycemic control (HbA1 < or = 10). Heated corn oil containing low or high levels of oxidized fatty acids was used as a test meal. At 2.5 h after the test meal, 50-ml blood samples were obtained from all subjects, and the chylomicron fraction (Sf > 1,000) was isolated. The degree of oxidation in chylomicrons was determined by measuring conjugated dienes. For determining the postprandial levels of triglycerides and of oxidized lipids in serum chylomicrons over an extended time period, blood samples were obtained at 0, 2.5, 5.0, and 7.5 h for isolation of chylomicrons and determination of fatty acid oxidation. RESULTS: We found that at 2.5 h after the consumption of the test meal containing either a low or high oxidized fatty acid content, conjugated dienes in serum chylomicrons in diabetic subjects in poor glycemic control were increased compared with those in control subjects. Diabetic patients in good glycemic control had similar levels of oxidized lipid in their chylomicrons when compared with control subjects. Additionally, in diabetic patients in poor glycemic control, the levels of oxidized lipids in chylomicrons remained elevated for an extended post-prandial period. CONCLUSIONS: In diabetic subjects with poor glycemic control, dietary oxidized lipids induce an exaggerated and sustained increase in the levels of oxidized lipids in chylomicrons when compared with either control subjects or diabetic patients with good glycemic control. These increased postprandial levels of potentially atherogenic oxidized lipids may contribute to the accelerated atherosclerosis associated with diabetes.

Blood Glucose↗
PubMed2003

Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling.

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys(396) and Cys(399)of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.

Amino Acid Motifs↗
PubMedDate not supplied

Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes?

Ram, fowl and bee spermatozoa, and oocytes of cows and zebrafish were used to study lipid membrane profiles, chilling sensitivity and lipid-phase transitions. The integrity of the membranes was determined by carboxyfluorescein diacetate (CFDA) staining following exposure for 15 minutes to low temperatures. Ram and fowl spermatozoa showed different degrees of loss of membrane integrity. Surprisingly, bee spermatozoa did not show any sensitivity to chilling, and their membranes remained intact down to 0 degree C. In bovine oocytes (at the GV stage) chilling injury was very severe at 16 degree C (membrane integrity decreased by 50%). Lipid phase transition (LPT) and membrane fluidity, which were evaluated by Fourier transform infrared (FTIR) microscopy, and fluorescence polarisation, showed phase transitions at the same temperatures as caused damage (between 30 and 12 degree C). The membrane lipid profiles showed high concentrations of polyunsaturated fatty acids (PUFA) in cold-sensitive ram spermatozoa and zebrafish oocytes, but the ratio between PUFA and saturated fatty acids was highest in cold-resistant bee spermatozoa and lowest in cold-sensitive bovine oocytes. These results suggest a close relationship among cold susceptibility, lipid phase transition and lipids profile in animal gametes.

Journal Article↗
PubMed1991

Intramolecular excimer formation of pyrene-labeled lipids in lamellar and inverted hexagonal phases of lipid mixtures containing unsaturated phosphatidylethanolamine.

The rates of intramolecular excimer formation of di(1'-pyrenemyristoyl)phosphatidylcholine (dipyPC) in dioleoylphosphatidylethanolamine (DOPE), egg PE/diolein (DG) and dilinoleoyl-PE (DLPE)/1-palmitoyl-2-oleoyl-PC (POPC) were studied at different temperatures and lipid compositions. Both the excimer-to-monomer intensity ratio and the excimer association rate constant were employed to quantify the rate of excimer formation. The latter was calculated from the measured monomer fluorescence lifetime of dipyPC. We observed that the rate of excimer formation was sensitive to either the temperature-induced or lipid composition-induced lamellar-to-inverted hexagonal phase transition of the above lipid systems. As the lipids entered the inverted hexagonal phase, the rate of excimer formation increased at the temperature-induced phase transition for DOPE, but decreased at the composition-induced phase transition for both TPE/DG and DLPE/POPC systems by increasing the DG% and decreasing the PC%, respectively. We conclude that the rate of intramolecular excimer formation of dipyPC in the non-lamellar phase is influenced both by the intra-lipid free volume of the hydrocarbon region and the intra-rotational dynamics of the two lipid acyl chains.

Journal Article↗
PubMed1990

Highly purified lipid X is devoid of immunostimulatory activity. Isolation and characterization of immunostimulating contaminants in a batch of synthetic lipid X.

Lipid X, an early precursor in the biosynthesis of lipid A has been reported to directly induce cytokine release in macrophages but also to inhibit endotoxin-induced tumor necrosis factor (TNF) induction. In this report we provide evidence that these conflicting results could be due to contaminants present in different batches of lipid X used. Thus, in an apparently pure batch of crystalline lipid X as obtained by a published procedure (Macher, I. (1987) Carbohydr. Res. 262, 79-84) small amounts of N,O-acylated disaccharide-1-phosphates could be identified. Their isolation was achieved by gel filtration on Sephadex LH-20 and further analysis of fractions showing elevated limulus amebocyte lysate values by thin layer chromatography and reverse-phase high performance liquid chromatography (HPLC) in combination with bioassays. Identification of immunostimulatory by-products was possible by testing HPLC-fractions for TNF-induction in bone marrow-derived mouse macrophages. Applying these procedures a disaccharide-1-phosphate, containing four 3(R)-hydroxymyristic acids at positions 2, 3, 2', 3', was identified as the main immunostimulatory side product. Two isomeric hydrolysis products of this compound with only three 3(R)-hydroxymyristic acid moieties attached to the disaccharide-1-phosphate were also identified. Surprisingly, these compounds behave quite differently in the TNF induction test. The disaccharide-1-phosphate, acylated at positions 2, 2', 3', is a very potent inducer of TNF-release whereas the corresponding isomer containing the 3(R)-hydroxymyristic acids in positions 2, 3, 2', does not induce TNF release, but strongly inhibits TNF release as induced by the former compound. Thus, contamination of "pure" lipid X with immunostimulatory or immunoinhibitory impurities may explain the divergent pharmacological profiles which were attributed to synthetic lipid X.

Acylation↗
PubMed1983

Enriched lipid diet accelerates lupus nephritis in NZB x W mice. Synergistic action of immune complexes and lipid in glomerular injury.

The effect of a lipid-rich diet was investigated in autoimmune NZB x W mice with immune complex lupus nephritis. Mice on an enriched lipid diet died sooner than controls. This increase in dietary fat elevated serum cholesterol levels and was associated with an accelerated loss of renal function. Ultrastructural examination of glomeruli revealed lipid in the peripheral loops and mesangium adjacent to immune deposits. Increased lipid accumulation was confirmed by histochemical staining and biochemical extraction. Accelerated disease was not related to changes in immune parameters. Thus, a high lipid consumption accelerated lupus nephritis by a local vascular effect and not by a systemic influence on the immune response. Therefore, in our system, a combined immunologic and lipid glomerular capillary insult caused a more rapidly progressive renal disease than immune complex injury alone.

Animals↗
PubMed1976

Lipid-saccharide intermediates in glycoprotein biosynthesis. II. Studies on the structure of an oligosaccharide-lipid from thyroid.

Structural studies have been performed on an oligosaccharide-lipid from thyroid believed to be an intermediate in glycoprotein synthesis. For these investigations the compound was isolated from the gland in unlabeled form as well as differentially radiolabeled in its saccharide, lipid, and phosphate portions by incubation of slices with [14C]- or [3H]glucose, [3H]mevalonic acid and [32P]phosphate, respectively. The unlabeled oligosaccharide-lipid was obtained in a chloroform/methanol/water (10/10/3) extract in a yield of about 1 nmol/g of thyroid and was purified therefrom by DEAE-cellulose chromatography. The saccharide moiety released from the glycolipid by mild acid hydrolysis was isolated by gel filtration and contained 11 mannose, 1 to 2 glucose, and 2 N-acetylglucosamine residues. The reducing terminal position of the oligosaccharide was occupied by 1 of the glucosamine residues and from these analyses a molecular weight of 2,415 was calculated. That glucose is an integral part of the molecule was further demonstrated by the finding that during Dowex 50 chromatography it remained as a constituent of the positively charged oligosaccharide produced by deacetylation with alkaline borohydride at 80 degrees. The phosphorus content of the purified unlabeled oligosaccharide-lipid was determined to be 2 residues per molecule, suggesting the presence of a pyrophosphate bridge between its carbohydrate and lipid portions. Further evidence for such a linkage region was provided by characterization of the products from mild acid and alkaline hydrolysis of the differentially radiolabeled glycolipid. These included dolichyl mono- and pyrophosphate, oligosaccharide phosphate, and free oligosaccharide. Digestion with alpha-mannosidase of the radiolabeled glycolipid led to the release of 39% of its mannose while from the free oligosaccharide 53% of this sugar was removed. Acetolysis of the [14C]oligosaccharide yielded a mannobiose and mannotriose as well as larger fragments consisting of mannose, glucose, and glucosamine. Smith periodate degradation gave rise to a small core segment (6 glycose residues) made up only of mannose and glucosamine from which half of the mannose residues could be released by alpha-mannosidase digestion. From these studies a tentative structure for the carbohydrate moiety of the oligosaccharide-lipid has been proposed. In this formulation an inner core (periodate-resistant) made up of 4 mannose and 2 N-acetylglucosamine residues is attached to the pyrophosphate group by the most internal glucosamine. This core, as well as an additional mannose and 1 to 2 glucose residues, constitutes the alpha-mannosidase-resistant fragment. More peripherally are found other mannose residues, all in alpha-linkage. In this structural scheme the glucose is located so as to prevent the enzymatic release of more internally situated alpha-linked mannose residues.

Acetylglucosamine↗
PubMed1986

Monoclonal antibodies recognizing lipid-laden cells and extracellular regions with lipid-deposits in atherosclerotic aorta.

Monoclonal antibodies against lipid-laden cells and against extracellular regions of lipid-deposits in atherosclerotic aorta were prepared. Mice were immunized with a delipidated homogenate of atherosclerotic aorta of Watanabe-heritable hyperlipidemic rabbits. Hybridomas were obtained by fusion and cultured in hypoxanthine, aminopterin and thymidine selection medium. Specific antibodies were selected by indirect immunohistochemical staining of frozen sections of atherosclerotic aorta. Nine clones that produced antibodies that stained the atherosclerotic intima exclusively were selected and cloned by limiting dilution. Finally two clones (FCR1a/201F, FCR1b/904B) producing antibodies specific to lipid-laden cells and one clone (EMR1a/212D) producing an antibody specific to regions with lipid deposits in the extracellular matrix were established. These monoclonal antibodies may help in understanding how lipids accumulate in atherosclerosis.

Animals↗